lv-max lentiviral production system · for research use only. not for use in diagnostic procedures....

For Research Use Only. Not for use in diagnostic procedures.

LV-MAX™ Lentiviral Production SystemUSER GUIDE

For suspension format lentiviral production in a chemically defined,serum-free medium

Catalog Number A35684Publication Number MAN0017000

Revision D.0

Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0017000

Revision Date DescriptionD.0 22 March 2019 Overhaul of the user guide to bring it up to current style and standards.

C.0 24 August 2018 Remove a related product

B.0 13 June 2018 Add flask type

A.0 14 July 2017 New document

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

©2019 Thermo Fisher Scientific Inc. All rights reserved.

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Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Components of the Gibco™ LV-MAX™ Lentiviral Production System . . . . . . . . . . . . . . . . . . . . 8Viral production cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Growth medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Supplement solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Transfection reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Enhancer solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ CHAPTER 2 Thaw and establish Gibco™ Viral Production Cells . . . . 10

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Count suspension cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Workflow: Thaw and establish Gibco™ Viral Production Cells . . . . . . . . . . . . . . . . . . . . . . . . 12

Thaw and establish Gibco™ Viral Production Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Cryopreserve Gibco™ Viral Production Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ CHAPTER 3 Produce lentiviral vector in a 2-mL 96-deepwell block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15


Optimized transfection conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Workflow: Lentiviral production in a 2-mL 96-deep well block . . . . . . . . . . . . . . . . . . . . . . . 16

Transfect cells and produce the lentiviral vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Prepare cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Dilute cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Make complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Transfect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Harvest the lentiviral vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Titer the lentiviral vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

LV-MAX™ Lentiviral Production System User Guide 3

■ CHAPTER 4 Produce lentiviral vector in a 50-mL conical tube . . . . . 19



Workflow: Lentiviral production in a 50-mL conical tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20


■ CHAPTER 5 Produce lentiviral vector in a shaker flask . . . . . . . . . . . . . 23

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Lentiviral production specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23


Workflow: Lentiviral production in a shaker flask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25


■ CHAPTER 6 Titer lentiviral vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Titer using GFP expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Perform lentiviral vector titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Calculate the lentiviral titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Titer using antibiotic selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Perform lentiviral vector titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Calculate the lentiviral titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

■ APPENDIX A Scaling up tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Tables for scaling up preset and custom sized production volumes . . . . . . . . . . . . . . . . . . . 35

Contents

4 LV-MAX™ Lentiviral Production System User Guide

■ APPENDIX B Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

■ APPENDIX D Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Contents


Product information

IMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.

Product description

The Gibco™ LV-MAX™ Lentiviral Production System is a high-yield lentiviralproduction system based on HEK293F cells adapted to a special chemically defined,serum-free and protein free LV-MAX™ Production Medium in suspension form.

The LV-MAX™ Lentiviral Production System provides cells, production medium,supplement, transfection reagent, and enhancer to produce high titer lentiviralvectors.

Contents and storage

Reagents provided in the kit are sufficient for 300 mL of lentiviral production volume.

Table 1 LV-MAX™Lentiviral Production System Starter Kit (Cat No. A35684)

Component Cat. No. Amount Storage

Viral Production Cells[1] (1 X 107 cells/mL) A35347 2 × 1 mL Liquid nitrogen[2]

LV-MAX™ Production Medium A3583401 1 L

• 2°C to 8°C

• Protectedfrom light

LV-MAX™ Transfection Kit

• LV-MAX™ Supplement

• LV-MAX™ Transfection Reagent

• LV-MAX™ Enhancer

A35346 1 Kit

• 15 mL

• 2 × 0.9 mL

• 12 mL

[1] In 90% LV-MAX™ Production Medium and 10% DMSO[2] Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at −80°C.

1


Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Table 2 Materials required for lentiviral vector production (all methods)

Item Source

CO2 resistant orbital shaker 88881101

125-mL shaker flasks for Viral Production Cells MLS

Adjustable micropipettors MLS

Laboratory mixer (Vortex mixer or equivalent) MLS

Equipment and reagents to determine cell density and viability MLS

LV-MAX™ Packaging Mix A43237

Opti-MEM™ I Reduced Serum Medium 31985088

For cryopreservation:

Sterile 0.2-μm filter MLS

Cryovials MLS

Table 3 Additional materials required for lentiviral vector production in a 96-deepwell block

Item Source

3 mm orbital shaker MLS

2-mL sterile 96-deep well block MLS

2-mL sterile 96-deep well block (V bottom) MLS

96-well round bottom plate MLS

Multi-channel micropipettors MLS

PureLink™ Air Porous Tape 12262010

Table 4 Additional materials required for lentiviral vector production in a 50-mLconical tube

Item Source

Nunc 50-mL conical tube 339653

Corning™ Mini Bioreactor Centrifuge Tube 07-202-150

50-mL conical tube holder MLS

Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile

Chapter 1 Product informationRequired materials not supplied 1


http://www.thermofisher.com

http://fisherscientific.com

Item Source

125 mL 4115-0125

250 mL 4115-0250

1 L 4115-1000

Table 5 Additional materials required for lentiviral vector production in a shaker flask

Item Source

Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile

125 mL 4115-0125

250 mL 4115-0250

1 L 4115-1000

Components of the Gibco™ LV-MAX™ Lentiviral Production System

The Gibco™ LV-MAX™ Lentiviral Production System is designed to produce high-titerlentiviral vectors using transient transfection of a high-density suspension cell systemcultured in chemically defined, serum-free medium.

The Gibco™ Viral Production Cells are a clonal derivative of the original HEK293F cellline, that have been adapted to suspension culture in LV-MAX™ Production Medium.These cells can be thawed directly into LV-MAX™ Production Medium.

Cell line characteristics:• Transformed via culture with sheared human adenovirus 5 DNA• Expresses E1A adenoviral gene• Lacks the SV40 large T antigen• Cell doubling time of ~26 hours• Achieves maximum cell densities of ~1 × 107 cells/mL in shaker flask cultures• High lentiviral production capabilities between cell passages 5–20

LV-MAX™ Production Medium is a complete, chemically defined, serum-free andprotein-free medium, developed for growth and transfection of Gibco™ ViralProduction Cells.

LV-MAX™ Supplement is a chemically defined, serum-free and protein-freeformulation designed to control cell growth during transfection and increase lentiviralvector production without compromising cell viability.

LV-MAX™ Transfection Reagent is uniquely designed for high efficiency co-transfection of multiple plasmids into high density Gibco™ Viral Production Cells,with low toxicity.

Viral productioncells

Growth medium

Supplementsolution

Transfectionreagent

Chapter 1 Product informationComponents of the Gibco™ LV-MAX™ Lentiviral Production System1


LV-MAX™ Enhancer is a chemically defined, serum-free, protein-free formulation thatis designed to boost lentiviral vector production in Gibco™ Viral Production Cells.

Enhancer solution

Chapter 1 Product informationComponents of the Gibco™ LV-MAX™ Lentiviral Production System 1


Thaw and establish Gibco™ ViralProduction Cells

Procedural guidelines

• All solutions and equipment that come in contact with cells must be sterile.• Thaw and transfer cells directly into a 125-mL shaker flask with 30 mL of pre-

warmed 37°C LV-MAX™ Production Medium. Centrifugation and mediumchange are not required when seeding the cells.

• Do not centrifuge the cells as this can lower cell viability.• Three days post-thaw, viable cell density should be approximately 1 × 106

cells/mL with ≥ 90% cell viability.• Subculture cells when they reach a density of ~3.5 to 5.5 x 106 viable cells/mL,

typically every 3–4 days.• Allow freshly thawed cells to recover in culture for 5 passages post-thaw before

using cells for virus production.• Inspect cells to ensure that round cell morphology is maintained over passages

for most of the cell population with minimal cell clumping. Proper cell phenotypeis critical for optimal transfection and virus production.

• Warm LV-MAX™ Production Medium to room temperature.For a faster warmup, place your medium at 37°C for no longer than 20 minutes.

• We recommend discarding cells after passage 20 from thaw.• The following table shows the recommended cell density for routine cell culture.

Subculture Seeding density

3 day 0.55 × 106 viable cells/mL

4 day 0.35 × 106 viable cells/mL

• The following table shows the recommended shaker speed and culture volumefor cultivation.

Production vessel Shaker speed(Orbit diameter) Vessel size

Cell culture volumerange (24%−40% of

vessel size)

Shaker flask(vented, notbaffled)

125 rpm (19 mm)120 rpm (25 mm)95 rpm (50 mm)

125 mL 30 mL − 50 mL

250 mL 60 mL − 100 mL

1 L 240 mL − 400 mL

2


Count suspension cells

Cell density is the critical driver of successful lentiviral production. The cell densitydepends on accurate cell counting. Follow these steps to ensure consistent andaccurate cell counts.

1. To minimize settling of cells after agitation, load the pipet aid in preparation.

2. Shake the flask in an orbital path,clockwise 3 times.

3. Shake the flask in an orbital path,counter clockwise 3 times.

4. Shake the flask back and forth threetimes, then left and right three times.

5. Pipet the cells up and down 3 times.

6. Remove 1 mL for cell counting, then count the cells.

7. Repeat cell counting at least 2 times to ensure accurate cell densities.Recount any cell counts with >10% variability to ensure a reliable average celldensity.

Chapter 2 Thaw and establish Gibco™ Viral Production CellsCount suspension cells 2


Workflow: Thaw and establish Gibco™ Viral Production Cells

Thaw cells

Cul

ture

cel

ls

Culture cells

Continue

P0

P1

P2

P3

P4

P5

P6

subculture

1. Day 1

3. Day 8

5. Day 15

7. Day 22

4. Day 12

6. Day 19

2. Day 4

Week 1

Week 2

Week 3

Week 4

Follow LV production protocol

8. Subculture and prepare cells for

next day LV production

Monday

Thursday

Monday

Friday

Monday

Friday

Monday

Chapter 2 Thaw and establish Gibco™ Viral Production CellsWorkflow: Thaw and establish Gibco™ Viral Production Cells2


Thaw and establish Gibco™ Viral Production Cells

Week 1: P0−P11. Day 1 Passage 0 (P0): Partially thaw a vial of Gibco™ Viral Production Cells in a

37°C water bath until only a small piece of ice remains (~2–3 minutes).

Note: We recommend starting on a Monday.

2. Transfer the contents of the vial into a 125-mL shaker flask containing 30 mL ofpre-warmed LV-MAX™ Production Medium.

Note: Centrifugation and medium change are not required for these cells.

3. Place the flask on an orbital shaker platform in an incubator set to 37°C, 8% CO2,and 80% relative humidity (for recommended shaker speeds, see Table 3).

4. Day 4 Passage 1 (P1): Seed cells at a density of 0.35 × 106 cells/mL in a totalculture volume of 40 mL in a new 125-mL shaker flask.

Week 2: P2−P35. Day 8 Passage 2 (P2): Seed cells at a density of 0.35 × 106 cells/mL in a total

culture volume of 40 mL in a new 125-mL shaker flask.

Note: By Passage 2, cell viability should be ≥90%.

6. Day 12 Passage 3 (P3): Seed cells at a density of 0.55 × 106 cells/mL in a totalculture volume of 40 mL in a new 125-mL shaker flask.

Note: Aliquot some cells for long-term storage, see “Cryopreserve Gibco™ ViralProduction Cells“ on page 14.

Week 3: P4−P57. Day 15 Passage 4 (P4): Seed cells at a density of 0.35 × 106 cells/mL in a total

culture volume of 75 mL in a new 250-mL shaker flask.

8. Day 19 Passage 5 (P5): Seed cells at a density of 0.55 × 106 cells/mL in 300 mL oftotal culture volume in a 1-L shaker flask to ensure appropriate cell numbers forlentivirus production.

Week 4: Cells are established and ready for lentivirus production9. Day 22 Continue to subculture cells when required until P20. For recommended

seeding densities for subculture, see List item. on page 10.

Proceed with lentiviral production.• For high throughput lentiviral production in a 2-mL 96-deep well block, proceed

to Chapter 3, “Produce lentiviral vector in a 2-mL 96-deep well block“.• For lentiviral production in a 50-mL conical tube, proceed to Chapter 4, “Produce

lentiviral vector in a 50-mL conical tube“.• For lentiviral production in a shaker flask, proceed to Chapter 5, “Produce

lentiviral vector in a shaker flask“.

Chapter 2 Thaw and establish Gibco™ Viral Production CellsThaw and establish Gibco™ Viral Production Cells 2


Cryopreserve Gibco™ Viral Production Cells

Use cells at passage 3 when cell density reaches 3.5−5.5 × 106 viable cells/mL and cellviability is >95%.

1. Prepare the appropriate amount of cryopreservation medium by combining LV-MAX™ Production Medium and DMSO according to the following table.

Reagent volume to add

LV-MAX™ Production Medium 9 mL

DMSO 1 mL

Total 10 mL

2. Mix well, then filter through a 0.2-µm filter.

3. Centrifuge the cells at 100 × g for 5 minutes to collect the contents at the bottom,then discard the supernatant.

4. Gently resuspend the cell pellet in cryopreservation medium.

5. Dilute the cells with cryopreservation medium to a final density of 1 × 107 viablecells/mL.

6. Aliquot 1 mL of cells per cryovial, then freeze the cells at −80°C for one day.

7. Transfer the frozen vials to liquid nitrogen for long-term storage.

Chapter 2 Thaw and establish Gibco™ Viral Production CellsCryopreserve Gibco™ Viral Production Cells2


Produce lentiviral vector in a 2-mL96-deep well block


• Follow the guidelines in Chapter 2, “Thaw and establish Gibco™ Viral ProductionCells“ to culture cells.

• Warm LV-MAX™ Production Medium to room temperature before transfection.• The LV-MAX™ Supplement, LV-MAX™ Transfection Reagent, and Opti-MEM™ I

Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vectors is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• Each well of the 2-mL 96-deep well block is appropriate for 1 mL of lentivirus

production.• The lentiviral production volume for each 96-deep well block is 105 mL, which

includes 10% overage.• Triplicate runs are recommended for each individual lentiviral transfer vector.

Optimized transfection conditions

Condition Amount

Total Opti-MEM™ I Reduced Serum Medium 2 × 5% of lentivirus production volume

Total DNA per mL of lentivirus production 2.5 μg/mL (LPM + LTV)

Ratio of lentiviral packaging mix (LPM) to lentiviraltransfer vector (LTV) 3 to 2

LV-MAX™ Transfection Reagent 6 µL/mL of lentivirus production volume

LV-MAX™ Enhancer 4% of the lentivirus production volume

Gibco™ Viral Production Cells 4 × 106 cells/mL production volume

Total LV-MAX™ Supplement 5% of lentivirus production volume

3


Workflow: Lentiviral production in a 2-mL 96-deep well block

1. Prepare cells

2. Plate cells

3. Make complex

4. Transfect cells

5. Add enhancer

7. Measure Titer

Day 0Monday

Day 1Tuesday

Day 3Thursday

24 - 28hr

1min

10min

5-6 hr

48 -55 hr

TfxRDNA

AB

CD

EF

GH

12

34

56

78

910 11 12

AB

CD

EF

GH

12

34

56

78

910 11 12

21

6. Harvest lentiviral vector

Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockWorkflow: Lentiviral production in a 2-mL 96-deep well block3


Transfect cells and produce the lentiviral vector

Note: This workflow guides you through the production of lentiviral vector from105 mL of production volume, which is enough for one 96-well block. For scaling uptables, see Appendix A, “Scaling up tables“.

1. Determine the cells/mL of the 3-day cultured high density cells.

2. Dilute the high density cells to 3.5 × 106 viable cells/mL, then subculture for afurther 24 hours.

1. Count the high density cells.

2. In a new sterile flask, add 4.20 × 108 cells to LV-MAX™ Production Medium tomake a final volume of 89.25 mL.

3. Add 5.25 mL of LV-MAX™ Supplement.

4. Mix the cells well by pipetting up and down, then transfer 900 µL to each well ofa 2-mL 96-deep well block.

5. Seal the 96-deep well block plate with PureLink™ Air Porous Tape, then place ona 3-mm orbital shaker at 1,250 rpm in an incubator.

1. Add 5.25 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 1: DNA.

2. Add 157.5 µg of packaging plasmid.

Note: The ratio of packaging plasmid to transfer plasmid is 3 to 2. For 2.5 µg/mLof total DNA, this translates to 1.5 µg of packaging plasmid to 1 µg of transferplasmid.

3. Pipet 50 µL into each well of a 96-well round bottom plate.

4. Add 1 µg of transfer plasmid to each well.

5. Add 5.25 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 2: TfxR.

6. Add 630 µL of LV-MAX™ Transfection Reagent (6 µL/mL).

7. Vortex briefly to mix, then incubate for 1 minute at room temperature.

8. Immediately pipet 50 µL into each well of the 96-well round bottom platecontaining the prepared DNA solution, then mix well by pipetting up and down.

9. Incubate the combined solution for 10 minutes at room temperature.

1. Add 100 µL of the DNA-lipid complex to each well of the previously prepared96-deep well block of cells.

Note: The DNA-lipid complex is stable for up to 1.5 hours.

Prepare cells

Dilute cells

Make complexes

Transfect cells

Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockTransfect cells and produce the lentiviral vector 3


2. At 5–6 hours posttransfection, add 40 µL of LV-MAX™ Enhancer to each well ofthe 96-deep well block.

Note: The optimum time frame for LV-MAX™ Enhancer addition is anytimebetween 5–14 hours posttransfection.

1. At 48–55 hours posttransfection, centrifuge the 96-deep well block at 900 × g in aswinging bucket centrifuge for 15 minutes.

2. Harvest the lentiviral vector by transferring the supernatant to a fresh 96-wellround bottom plate.

Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multiple96-well round bottom plates.

3. Immediately following harvest, store the lentiviral vector at -80°C.

Thaw, then titer the lentiviral vector using your method of choice.

Note: Thaw the virus on ice before use.

For recommended protocols, see Chapter 6, “Titer lentiviral vector“.You can optimize your lentiviral vector yield by trying different starting cell densities,such as 2.5, 3, and 4 × 106 viable cells/mL.

Harvest thelentiviral vector

Titer the lentiviralvector

Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockTransfect cells and produce the lentiviral vector3


Produce lentiviral vector in a 50-mLconical tube




Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vectors is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• This protocol is appropriate for lentiviral production volumes of 5–30 mL.• Fix a 50-mL conical tube holder onto an orbital shaker in an incubator. Set the 19-

mm orbital shaker speed to 250 rpm.


Condition Amount








4


Workflow: Lentiviral production in a 50-mL conical tube

2. Dilute cells

3. Make complex

4. Transfect cells

5. Add enhancer

7. Measure Titer

24-28hr

1min

10min

5-14 hr

48-55 hr

TfxR DNA

AB

CD

EF

GH

12

34

56

78

910 11 12

2 1

Day 0Monday

Day 1Tuesday

Day 3Thursday

1. Prepare cells


Chapter 4 Produce lentiviral vector in a 50-mL conical tubeWorkflow: Lentiviral production in a 50-mL conical tube4



Note: This workflow guides you through the production of lentiviral vector from10 mL of production volume, in one 50-mL conical tube. For scaling up tables, see Appendix A, “Scaling up tables“.




2. In a new 50-mL conical tube, add 4 × 107 cells to LV-MAX™ Production Mediumto make a final volume of 8.5 mL.


4. Mix the cells well by pipetting up and down, then place on a 19-mm orbitalshaker at 250 rpm in an incubator.

5. Use a mini bioreactor cap to seal the top of the conical tube in place of theoriginal cap.

1. Add 0.5 mL of Opti-MEM™ I Reduced Serum Medium to a 1.5-mLmicrocentrifuge tube and label it Tube 1: DNA.


2. Add 15 µg of packaging plasmid.

3. Add 10 µg of transfer plasmid, then vortex briefly to mix.

4. Add 0.5 mL of Opti-MEM™ I Reduced Serum Medium to a 1.5-mLmicrocentrifuge tube and label it Tube 2: TfxR.



7. Combine Tube 1:DNA with Tube 2:TfxR, then mix well by pipetting up anddown.


1. Add 1 mL of the DNA-lipid complex to the previously prepared tube of cells,then mix well by pipetting up and down gently.


Prepare cells

Dilute cells

Make complexes

Transfect cells

Chapter 4 Produce lentiviral vector in a 50-mL conical tubeTransfect cells and produce the lentiviral vector 4


2. At 5–6 hours posttransfection, add 0.4 mL of LV-MAX™ Enhancer.


1. At 48–55 hours posttransfection, collect the culture medium, then centrifuge thecells at 1,300 × g in a swinging bucket centrifuge for 15 minutes.For smaller volumes, use a 1.5-mL microcentrifuge tube and centrifuge in abenchtop centrifuge at 13,000 rpm for 5 minutes.

2. Harvest the lentiviral vector by transferring the supernatant to a fresh tube.

3. Further remove cell debris by filtration through a 0.45 µm low protein bindingfilter.


Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multipletubes.






Chapter 4 Produce lentiviral vector in a 50-mL conical tubeTransfect cells and produce the lentiviral vector4


Produce lentiviral vector in a shakerflask




Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vector is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• This protocol is appropriate for lentiviral production volumes of 30–500 mL.

Production vessel Shake speed(orbital diameter) Vessel size

LV production volumerange (24%−50%[1] of

vessel size)

Shaker flask(vented, no baffle)

125 rpm (19 mm)

120 rpm (25 mm)

95 rpm (50 mm)

125 mL 30−62.5 mL

250 mL 60−125 mL

500 mL 120−250 mL

1,000 mL 240−500 mL

2,000 mL 480−1,000 mL

[1] LV production volume can be up to 54% of vessel size, including LV-MAX™ Enhancer. Do not exceed 60%.


Condition Amount





5

Lentiviralproductionspecifications


Condition Amount




Chapter 5 Produce lentiviral vector in a shaker flaskOptimized transfection conditions5


Workflow: Lentiviral production in a shaker flask

1. Prepare cells

2. Dilute cells

3. Make complex

4. Transfect cells

5. Add enhancer

7. Measure Titer

24-28hr

1min

10min

5-14 hr

48-55hr

TfxR DNA

12

AB

CD

EF

GH

12

34

56

78

910 11 12

Day 0Monday

Day 1Tuesday

Day 3Thursday


Chapter 5 Produce lentiviral vector in a shaker flaskWorkflow: Lentiviral production in a shaker flask 5



Note: This workflow guides you through the production of lentiviral vector from30 mL of production volume, in one 125-mL shaker flask. For scaling up tables, see Appendix A, “Scaling up tables“.




2. In a new 125-mL shaker flask, add 1.2 × 108 cells to LV-MAX™ ProductionMedium to make a final volume of 25.5 mL.


4. Mix the cells well by pipetting up and down, then place on a 19-mm orbitalshaker at 125 rpm in an incubator.

1. Add 1.5 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 1: DNA.


2. Add 45 µg of packaging plasmid.

3. Add 30 µg of transfer plasmid, then vortex briefly to mix.

4. Add 1.5 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 2:TfxR.



7. Combine Tube 1:DNA with Tube 2:TfxR, then mix well by pipetting up anddown.


1. Add 3 mL of the DNA-lipid complex to the previously prepared flask of cells,then mix well shaking gently.


2. At 5–6 hours posttransfection, add 1.2 mL of LV-MAX™ Enhancer.


Prepare cells

Dilute cells

Make complexes

Transfect cells

Chapter 5 Produce lentiviral vector in a shaker flaskTransfect cells and produce the lentiviral vector5


1. At 48–55 hours posttransfection, collect the culture medium, then centrifuge thecells at 1,300 × g in a swinging bucket centrifuge for 15 minutes.For smaller volumes, use a 1.5-mL microcentrifuge tube and centrifuge in abenchtop centrifuge at 13,000 rpm for 5 minutes.

2. Harvest the lentiviral vector by transferring the supernatant to a fresh tube.

3. Further remove cell debris by filtration through a 0.45 µm low protein bindingfilter.


Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multipletubes.






Chapter 5 Produce lentiviral vector in a shaker flaskTransfect cells and produce the lentiviral vector 5


Titer lentiviral vector

IMPORTANT! Handling of lentivirus must be performed as per institutionalguidelines. All materials should be treated with a 10% bleach solution prior todisposal.

Titer using GFP expression

• Thaw the virus on ice before use. Do not use any acceleration method because itcan decrease the virus titer.

• Mix the virus by tapping or inverting the tube. Do not vortex and avoid mixingvigorously.


Item Source

HT1080 cell line ATCC, CCL-121

DMEM, high glucose, GlutaMAX™ Supplement, pyruvate[1] 10569010

Polybrene [2] Fisher Scientific™,NC0663391

96-well culture plates MLS

96-well round bottom plates MLS

Large swinging bucket centrifuge MLS

TrypLE™ Express Enzyme (1X), no phenol red 12604013

DPBS, no calcium, no magnesium 14190250

[1] Add 10% FBS to make culture medium for HT1080 [2] Stock solution: 10 mg/mL in sterile H2O

Day 1 (morning) - infect HT1080 cells

1. Four hours before infection, seed a 96-well culture plate according to thefollowing table:

Cells Density Volume of media

HT1080 7,000 cells/well 100 µL/well

Note: Cells are ~30% confluent at time of infection.

6

Proceduralguidelines

Requiredmaterials notsupplied

Perform lentiviralvector titration




2. Two hours before infection, thaw the virus on ice.

Note: Do not accelerate the lentiviral vector thawing process because it candecrease the titer of the virus.

3. Prepare dilution medium by combining 15 mL of fresh culture medium with12 µL of 10 mg/mL Polybrene (final concentration of 8 µg/mL), then vortex tomix.

4. Prepare a 4-log serial dilution (10-1 to 10-4) by sequentially diluting 15 µL of virusinto 135 µL of dilution medium per well of a round bottom 96-well plate.

Note: We recommend performing each dilution in quadruplicate.

Note: If your virus was concentrated, you may need more dilutions.

5. Remove the culture media from the HT1080 cells, then infect by transferring100 µL of the diluted virus to each corresponding well.

6. Centrifuge the infected cell plate at 900 × g for 30 minutes at room temperature.

7. Incubate the infected cell plate overnight.

Day 2 (morning) – change medium, then incubate cells

1. Remove the medium from each well, then add 100 µL/well of fresh HT1080culture medium (without Polybrene).

Note: When replacing medium, always start from the Negative Control andwork backwards to minimize the amount of virus carried over.

2. Incubate the infected plate for 72 hours.

Day 5 - prepare cells, then run through a flow cytometer

1. Remove the medium from each well, then add 150 µL of trypsinization medium(75% TrypLE™ + 25% DPBS mixture) to each well.

2. Incubate the infected plate for 15 minutes in an incubator at 37°C.

3. Use a microscope to check the cells ands ensure that the cells are rounded up.

4. Detach the cells from the culture plate bottom by gently pipetting up and down.

5. Process the cells through a flow cytometer.

Chapter 6 Titer lentiviral vectorTiter using GFP expression 6


1. To calculate the titer (Transforming Units/mL), determine the appropriatedilution factor to use based on the % of GFP+ cells.The optimal infection range is 1−20% of GFP+ cells. Over 20% of GFP+ cellsresults in an underestimated lentiviral vector titer.

Lentivirus dilution EmGFP+ cells

102 96%

103 65%

104 18%

Using this table of results, the 104 dilution is selected for the calculation becausethe % of GFP+ cells falls in the desired 1−20% range.

2. Use the following formula to calculate the titer:Titer = (F × C/V) × D

• F = frequency of GFP+ cells (%GFP+ cells/100)• C = cell number per well at the time of transduction (7,000 cells)• V = volume of inoculum in mL (0.1 mL)• D = lentivirus dilution factor

Example:• F = 18/100 (using the table in Step 1)• C = 7,000 (cell number at the time of infection)• V = 0.1 (100 µL of medium)• D = 104

The calculation is:Titer = (0.18 × 7,000/0.1) × 104 = 1.26 × 108 TU/mL

Calculate thelentiviral titer

Chapter 6 Titer lentiviral vectorTiter using GFP expression6


Titer using antibiotic selection

• Thaw the virus on ice before use. Do not use any acceleration method because itcan decrease the virus titer.

• Mix the virus by tapping or inverting the tube. Do not vortex and avoid mixingvigorously.


Item Source

HT1080 cell line ATCC, CCL-121

DMEM, high glucose, GlutaMAX™ Supplement, pyruvate[1] 10569010

Polybrene[2] Fisher Scientific™,NC0663391

Crystal violet Fisher Scientific™,ICN15251150

Blasticidin S HCl[3] A1113903

Puomycin[4] A1113802

6-well culture plates 140675

TrypLE™ Express Enzyme (1X), no phenol red 12604013

[1] Add 10% FBS to make culture medium for HT1080 [2] Stock solution: 10 mg/mL in sterile H2O and filter through 0.2 µm filter[3] Selection medium: culture medium with Blasticidin S HCl, 10 μg/mL final concentration (the selection

medium based on pLenti transfer plasmid back bone selection marker)[4] Select culture medium final concentration 10 μg/mL

Day 1 (morning) – infect HT1080 cells

1. Four hours before infecting cells, seed a 6-well plate according to the followingtable:

Cells Density Volume of media

HT1080 210,000 cells/well 2 mL/well

Note: Cells are ~30% confluent at time of infection.

2. Two hours before infection, thaw the virus on ice.

Note: Do not accelerate the lentiviral vector thawing process because it candecrease the titer of the virus.

3. Prepare dilution medium by combining 20 mL of fresh culture medium with16 µL of 10 mg/mL Polybrene (final concentration of 8 µg/mL), then vortex tomix.

Proceduralguidelines

Requiredmaterials notsupplied

Perform lentiviralvector titration

Chapter 6 Titer lentiviral vectorTiter using antibiotic selection 6




4. For a virus sample, prepare seven microcentrifuge tubes and label them as T1, T2to T7.

5. Add 180 µL of prepared dilution medium to T1, then add 900 µL to T2 throughT7.

6. Add 20 µL of virus sample, crude lentivirus, or concentrated virus aliquot to T1(101 dilution), then invert the tube to mix.

T1 T2 T3 T4 T5 T6 T7

100 µL

180 µL

10 10 10 10 10 10

900 µL 900 µL 900 µL 900 µL

20 µL

Medium

Dilution

LVVs

100 µL 100 µL 100 µL 100 µL 100 µL

2 3 4 5 6 107

900 µL900 µL

7. Use a new tip to transfer 100 µL of T1 dilution to T2, then invert the tube to mix(102 dilution).

8. Use a new tip to transfer 100 µL of T2 dilution to T3, then invert the tube to mix(103 dilution).

9. Repeat Step 8 to complete dilutions T4 to T7.

Note: If your virus was concentrated, you may need more dilutions.

10. Replace the culture medium in A1, A2, A3, B1, and B2 of the prepared 6-wellplate of HT1080 cells with 1,500 µL of prepared dilution medium, then 2,000 µLto B3 as a negative control (‘Neg Ctrl’)

11. Pipet 500 µL of diluted virus into each respective well, starting with T3,according to the following figure.

LVVs infection grid

T3A

1 2 3

3

B

500 µLT4

500 µLT5

4 x10

64 x10 74 x10

44 x10

CtrlNeg

54 x10500 µL

T6500 µL

T7500 µL

Final viral dilution

T3A

1 2 3

B

T4 T5

T6 T7CtrlNeg

12. Ensure even distribution of the diluted virus across each well by moving theinfected 6-well plate left, right, backward, then forward several times.

Chapter 6 Titer lentiviral vectorTiter using antibiotic selection6


13. Centrifuge the infected cell plate at 900 × g (using a swinging bucket centrifuge)at room temperature for 30 minutes.

14. Incubate the infected cell plate overnight.

Day 2 (morning) – change medium, then incubate cells

1. Remove the medium from each well, then add 2 mL/well of fresh HT1080 culturemedium (without Polybrene).

Note: When replacing medium, always start from the Negative Control andwork backwards to minimize the amount of virus carried over.

2. Incubate cells for 24 hours.

Day 4 – start antibiotic selection

1. Prepare HT1080 culture medium with antibiotic, such as Blasticidin with a finalconcentration of 10 µg/mL or Puromycin with a final concentration of 1 µg/mL.

Note: Select antibiotic based on your pLenti-transfer back bone selection marker.

2. Replace the culture medium in each well with 2 mL of prepared selectionmedium, then incubate for 48 hours.

3. Repeat Step 2 until the ‘NegCtrl’ well has no cells left.This is an approximately 10-day process.

Day 13 – stain with Crystal Violet

1. Prepare the Crystal Violet staining solution: 1% Crystal Violet dissolved in a 10%ethanol/H20 solution.

2. Remove the antibiotic selection medium from the 6-well plate, then wash eachwell with 2 mL of PBS.

3. Add 1 mL of prepared Crystal Violet staining solution to each well, then incubatethe plate for 20 minutes at room temperature.

4. After 20-minutes incubation, remove the staining solution from the 6-well plate.Store the staining solution because it can be reused.

5. Wash the stained 6-well plate with water several times until a clear backgroundappears.

1. Visually count the number of stained colonies per well.

2. Use the following formula to calculate the titer (Transforming Units/mL) of thevirus stock:Titer = (Number of discrete colonies × dilution factor) / Volume of inoculumExample:Count = 30 colonies stained in the 4 × 106 dilution well (B-1) and 5 coloniesstained in the 4 × 107 dilution well (B-2)Volume of diluted virus = 2 mL

Calculate thelentiviral titer

Chapter 6 Titer lentiviral vectorTiter using antibiotic selection 6


The calculation is:Titer = (30 × 4 × 106 + 5 × 4 × 107)/2/2 = 8 × 107 TU/mL

Chapter 6 Titer lentiviral vectorTiter using antibiotic selection6


Scaling up tables

Tables for scaling up preset and custom sized production volumes

This section contains two tables for use in scaling up the production volume.

The first table (Table 6 on page 36) displays all the required volumes for each reagent based on the presetproduction vessel sizes that can be used.

The second table (Table 7 on page 37) displays the formula for each of the required volumes for each reagentbased on the custom production volume to be used. Use the formula in the table for each reagent to calculateyour specific volume, then input that volume in the space provided under the correct heading.

An explanation of the scaling up table headings are in the following list:• Production vessel: The vessel used for culturing the lentiviral vector (ensure the vessel size is large enough

for the production volume plus all other reagents).• Production volume: This is the initial volume of media and cells combined.• Number of cells: The number of cells required.• Volume of media: The amount of media to dilute the cells in.• Supplement: The volume of LV-MAX™ Supplement required.• Opti-MEM I: The volume of Opti-MEM™ I Reduced Serum Medium required for tube 1 (DNA).• Packaging: The amount of packaging plasmid required for tube 1 (DNA).• Transfer: The amount of transfer plasmid required for tube 1 (DNA).• Opti-MEM I: The volume of Opti-MEM™ I Reduced Serum Medium required for tube 2 (TfxR).• LV-MAX™ Transfection Reagent: The amount of LV-MAX™ Transfection Reagent required for tube 2 (TfxR).• Enhancer: The amount of LV-MAX™ Enhancer required.

A


Table 6 Preset scaling up

Productionvessel

Productionvolume

Diluted cell preparation Tube 1 (DNA) Tube 2 (TfxR)

LV-MAX™

EnhancerNumber ofcells

Volume ofmedia

LV-MAX™

Supplement

Opti-MEM™ I

ReducedSerum

Medium

Plasmids[1] Opti-MEM™ I

ReducedSerum

Medium

LV-MAX™

TransfectionReagentPackaging Transfer

96 deepwell plate 1 mL 4 × 106

0.85 mL −volume of3-day culturedcells

50 µL 50 µL 1.5 µg 1 µg 50 µL 6 µL 40 µL

50-mLconical 10 mL 4 × 107


500 µL 500 µL 15 µg 10 µg 500 µL 60 µL 400 µL

125-mLshakerflask

30 mL 1.2 × 108


1.5 mL 1.5 mL 45 µg 30 µg 1.5 mL 180 µL 1.2 mL

1-Litershakerflask

400 mL 1.6 × 109

340 mL −volume of3-day culturedcells

20 mL 20 mL 600 µg 400 µg 20 mL 2.4 mL 16 mL

2-Litershakerflask

1 L 4 × 109

850 mL −volume of3-day culturedcells

50 mL 50 mL 1.5 mg 1 mg 50 mL 6 mL 40 mL

[1] The ratio of packaging plasmid to transfer plasmid is 3:2. The final concentration of packaging plasmid is 2.5 µg/mL of production volume.

Appendix A

Scaling up tablesTables for scaling up preset and custom

sized production volumes

A36LV-M

AX™ Lentiviral Production System

User G

uide

Table 7 Custom scale up

Productionvessel

Productionvolume

Diluted cell preparation Tube 1 (DNA) Tube 2 (TfxR)

LV-MAX™

EnhancerNumber ofcells

Volume ofmedia

LV-MAX™

Supplement

Opti-MEM™ I

ReducedSerum

Medium

Plasmids[1] Opti-MEM™ I

ReducedSerum

Medium

LV-MAX™

TransfectionReagentPackaging Transfer

96 deepwell plate 1 mL 4 × 106


50 µL 50 µL 1.5 µg 1 µg 50 µL 6 µL 40 µL

Desiredvessel n mL

n × number of

cells

n × Volume of

media

n × LV-MAX™

Supplement

n × Opti-

MEM™ IReducedSerum

Medium

n × Packaging

n × Transfer

n × Opti-

MEM™ IReducedSerum

Medium

n × LV-MAX™

TransfectionReagent

n × LV-MAX™

Enhancer

Desiredvessel n × 1 mL n × 4 × 106

n × (0.85 mL −volume of3-day culturedcells)

n × 50 µL n × 50 µL n × 1.5 µg n × 1 µg n × 50 µL n × 6 µL n × 40 µL

[1] The ratio of packaging plasmid to transfer plasmid is 3:2. The final concentration of packaging plasmid is 2.5 µg/mL of production volume.

Appendix A

Scaling up tablesTables for scaling up preset and custom

sized production volumes

A

LV-MAX

™ Lentiviral Production System U

ser Guide

37

Related products

Related products

Unless otherwise indicated, all materials are available through thermofisher.com.

Item Source

Vivid Colors™ pLenti6.3/V5-GW/EmGFP Expression Control Vector V37006

Countess™ II Automated Cell Counter AMQAX1000

Large capacity cell culture incubator controls 37°C, 8% CO2 and75−80% humidity

3950 (if applicablefor scale-up)

B



Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, and so on). To obtain SDSs, see the “Documentationand Support” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix C SafetyChemical safetyC


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safety C


https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.• Worldwide contact telephone numbers• Product support information

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• Order and web support• Product documentation

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you haveany questions, please contact Life Technologies at www.thermofisher.com/support.

D


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