Experiment of cytobiology

schedual

• cell culture and counting cell• isolation of subcellular structure• cell proliferation assays• methods in apoptosis

• Finish the experiment• Finish the experiment report • Clean the lab after experiment

Cell cultureand

Counting cell

Cell culture

cell culture

• Many research need the use of animal cells as in vitro model system.

• 1st cell line, Hela, from human cervical cancer.

• culture cells taken directly from animal tissues or organs are primary culture cell ( primary culture ).

• culture cells taken from primary culture cell are subculture cell (subculture)

• Growing in suspending or growing as adherent monolayer attached to the surface of tissue culture flask. Most culture cell are latter.

• culture cells should be maintained under mimic culture conditions as in the body

• must sterile• Need nutrient culture medium and

providing optimal temperature and PH(7.2-7.4, by sodium bicarbonate)

Material and instrument needed

• Biosafety cabinet (Laminar flow cabinet)

• Incubator with CO2 • 5% CO2

• nonvented tissue culture flasks (with lid)

• dropping pipette

• inverted microscope

• Liquid nitrogen storage vessel

culture medium needed

• Formulations are vary according to the type of the cell cultured.

• RPMI 1640, MEM, DMEM• Serum (10%, from newborn bovine)

• Phenol red needed as it will turn purple above PH7.6 and yellow below PH7.0.

Examine the condition of the cell (today)• using inverted microscope• at 10x objective • the clarity and color of culture solution• density and shape of the culture cells• ensure the cells are healthy and sub conflue

nt and free of contamination

光学显微镜（ 10 倍）下活细胞形态

光学显微镜（ 40 倍）下活细胞形态

HeLa

细胞呈圆形，边缘清晰整齐，贴壁生长

细胞形态不规则，向四周伸展伪足，边缘不清，贴壁生长

Material and instrument (today)

• Biosafety cabinet • CO2 Incubator• inverted microscope• Subculture cell （ CHL ）• nonvented tissue culture flasks (with lid), drop

ping pipette• Culture medium ： RPMI 1640 with 10% newb

orn bovine serum • 0.25% trypase solution （ pH7.2 ）

procedure

• Subculture cell (CHL) → discard origin culture medium solution → dealed with trypase solution for 30 sec → abandon trypase solution → then add 2ml culture medium solution → produce cell suspension (mix by pipette up/down several times), keep 1 drop of solution for counting → divide a half of cell suspension to another culture flask → add culture medium solution both flasks → incubate at 37℃ → observe after 2 days.

Before (left) and after (right) dealed with trypase solution

work in this order

• clean hands with alchole• work close to the flame (ignite at start)• put the culture flasks upslope

Counting cell

counting cell

• For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration.

counting cell

• The number of cells per milliliter of medium or per centimeter squared area of attach surface

Material and instrument

• CHL cell• 0.25% trypase solution （ pH7.2 ）• Culture medium solution

Material and instrument

• Hemocytometer is used for determining the number of cells per unit volume of a suspension

• Optical microscope

• Hemocytometer has 2 counting chambers

• Each chamber has 9 squares (subgrids), each square being 1mm2

procedure

• Subculture cell (CHL) → discard origin culture medium solution → dealed with trypase solution for 30 sec → abandon trypase solution → then add 2ml culture medium solution → produce cell suspension → counting at 10x (10x objective)

The use of Hemocytometer

• Hemocytometer has a glass coverslip of precise thickness which is supported 0.1mm above the ruled area.

• This coverslip is specially made and thicker since they must be heavy enough to overcome the surface tension of a drop of liquid.

• It should be placed over the counting surface prior to putting on the cell suspension.

• The suspension is introduced into one of counting chambers with a pipet.

• The area under the coverslip fills by capillary action.

• Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.

• The volume of liquid over each subgrid is 0.1mm3

• Count 4 corner squares, start in the upper left small square and follow the pattern

CalculationC=N × 104×DC: cells per mlN: the average number of cells counted in e

ach corner square （４ squares÷4 ）D: the dilution factor 1ml ＝ 1000mm3

Count the fields in this order

• Count all of the cells within each square, including cells touching the lines at the top and on the left. Do not count any cells that touch the lines on the right or at the bottom.

• Do not damage the coverslide• It is improperly loaded with flooded or

with air bubbles

The end