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Lipodin-Pro™ExperimentalResults

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Lipodin-ProTM - Protein Transfection

Reagent

Description Thedeliveryofproteins inside livingcellsrepresentsanalternativetonucleicacidstransfectionandapowerfulstrategy for functional studies or therapeutic approaches. Several technologies based on the use of peptidetransduction domain (PTD) were developed successfully to transduce proteins across the plasma membrane.However, these PTD poorly interactwith proteins, and covalent linkage between the protein and PTD ismostoften required. Lipodin-Pro™ is a formulation of lipids able to capture proteins through electrostatic andhydrophobicinteractions.Thereisnoneedforcovalentlinkageprocedure,Lipodin-Pro™isdirectlymixedwiththeproteinofinterestfor10minutes.Themixtureisthenaddedtothecellsinculture,thelipid-proteincomplexesare internalized by the cells and the proteins are released into the cytoplasm within few hours without anycytotoxicity. The optimized formulation of Lipodin-Pro™ is fully biodegradable maintaining a high cell viabilityupondelivery.Theproteinsdelivered insidethecellswithLipodin-Pro™retainboththeirstructureandfunctionwhetherpeptides,proteinsorantibodiesareused.

1. KitBenefitsLipodin-Pro™ can be used in various functional studies for cell signaling and apoptotic assays, protein-proteininteraction,proteinlocalizationandcompartmentshuttling.Whentheproteinisconjugatedtoafluorescentdye,thefunctionalassaycanbecarriedoutinlivingcellsundermultipletreatmentswithasinglesample.

PrincipalLipodin-Pro™advantages:

• NoneedforDNAcloningornucleicacidtransfection• Nochemicalligationorcrosslinking• Serumcompatible,nocytotoxicityandbiodegradable• Easier2-stepprotocolwithready-to-usereagents• Deliverfunctionallyactiveproteinwithinhours• Higherdeliveryefficiencywithstablecelllinesandprimarycells



2.TransfectedProteinsLipodin-Pro™successfullydeliverednumerousproteinsinawidevarietyofcells:

• BandR-phycoerythrin• bovineserumalbumin• β-galactosidase• humanactivecaspase-3,caspase8andcaspase9• immunoglobulins(unconjugatedorlabeledwithFITC,TRITC,AlexaFluor®488andAlexaFluor®546)• MBP-fusionprotein

Impurities,contaminantsandadditivespresentwiththeproteinofinterestaffectdeliveryefficiency.Werecommendusingaproteinsampleaspureaspossible.Wenoticedthatproteinpreparationscontaininghighcontentsofdetergentsorsodiumazidewerenotcompatiblewithproteintransfection,whereasglycerolhasnoeffect.



3.CellTypesTestedLipodin-Pro™ is applicable on numerous cell types. This reagent has been successfully tested on a variety ofimmortalized cell lines as well as primary cells (Table 1). By submitting end-user data to our TechnicalDepartment,wecanupdatethislistandimproveourtechnicalsupporttothescientificcommunity.Ifaparticularcell type isnot listed, thisdoesnot imply thatLipodin-Pro™ isnotworking for that typebut that ithasnotyetbeentested.

Table1:ExampleofcellssuccessfullytestedwithLipodin-Pro™reagent.

CellLine CellType Source Efficiency3T6 Embryonicfibroblasts Mouse 50%A549 Non-smallcelllungcarcinoma Human 50-80%B16-F10 Melanoma Mouse 50%BEAS-2B Bronchialepithelialcells Human 80%BHK21 Fibroblasts(Kidney) Hamster 80-90%CHO-K1 Epithelial-like(Ovary) Hamster 50-80%COS-1,COS-7 Fibroblasts(Kidney) GreenMonkey 50-70%HaCaT Keratinocytes Human 50-80%HEK-293 TransformedEmbryonic(Kidney) Human 80-100%HeLa CervicalEpithelialCarcinoma Human 50-60%Jurkat Tcellleukemia Human 50%L929 Fibrosarcoma Mouse 80-90%K562 Myelogenousleukemia Human 10-50%MDCK Epithelial(Kidney) Canine 10-50%N2A Neuroblastoma Mouse 60-80%NIH3T3 Fibroblasts Mouse 50-75%Raw264.7 Monocytes/macrophages Mouse 90%U87 Glioblastoma Human 50%Vero10A1 Epithelial(Kidney) Monkey 50%Primarycells Neurons Rat 50%Glialcells Rat 50%

4.DeliveryofBandR-Phycoerythrin

NIH3T3 A549



RAW 264.7 BHK21

B-Phycoerythrin(1µg,Sigma-Aldrich)wasdeliveredintheindicatedcell lineswith2µlofLipodin-Pro™reagent.Phycoerythrin/Lipodin-Pro™complexeswere incubated for24hours in24-wellplatesbeforeunfixedcellswereobservedbyfluorescencemicroscopy.

BEAS-2B BEAS-2B

HeLa 3T6



Vero Raw 264.7

R-Phycoerythrin (1 µg, Molecular Probes) was delivered in the indicated cell lines with 2 µl of Lipodin-Pro™

reagent. Phycoerythrin/Lipodin-Pro™ complexes were incubated for 24 hours in 24-well plates before unfixedcellswereobservedbyfluorescencemicroscopy.

5. DeliveryofBeta-Galactosidase

HeLa CHO-K1

A549 A549



β-Galactosidase(1µg)wasdeliveredinvariouscellswith2µlofLipodin-Pro™reagent.β-Galactosidase/Lipodin-Pro™ complexes were incubated for 24 hours in 24-well plates before fixed cells were stained for beta-galactosidaseactivity.

6.DeliveryofBSA-TRITC

BEAS-2B NIH3T3

Jurkat BHK21

Tetramethylrhodamine-labeled BSA (BSA-TRITC, 2 µg) was delivered in various cells with 3 µl of Lipodin-Pro™

reagent. BSA-TRITC/Lipodin-Pro™ complexes were incubated for 24 hours with cells in 24-well plates beforeunfixedcellswereobservedwithafluorescentmicroscope.



7.DeliveryofMBPFusionProtein

MBP-Proteinalone MBP-ProteindeliverywiththeLipodin-Pro™

reagent

TheMBP-fusionprotein(10µg)wasdeliveredwith25µlofLipodin-Pro™reagentinHEK293cells.Afteran8-hourincubation, cellswere fixed and immunostainedwith an anti-MBP antibody. Then, the cellswere observed byfluorescencemicroscopy.Aftera10-hourincubation,cellswerelysedforproteindetectionbywestern-blot.

8. DeliveryofImmunoglobulins

A Nuclear Pore Complex Protein antibodylabeled with AlexaFluor®488 (0.5 µg) wasmixed with 2 µl of Lipodin-Pro™ reagent andincubated for24hourswithBEAS-2Bcells ina24-well plate. Then, cells were fixed with 2%pFAandobservedbyfluorescencemicroscopy.

A control antibody labeled withAlexaFluor®488(0.5µg)wasmixedwith2µlofLipodin-Pro™ reagent and incubated for 24hourswithBHK-21cellsin24-wellplates.Then,cellswere fixedwith2%pFAandobservedbyfluorescencemicroscopy.

MBP-ProteinLipodin-Pro


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9. KineticsofR-PhycoerythrinDeliveryinNIH3T3Cells

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2h 4h 24h

R-Phycoerythrin(1µg)wasdeliveredinNIH3T3cellswith2µlofLipodin-Pro™reagentin24-wellplates.Cellswerecollected and fixed with 2% PFA at the indicated time point. The number of fluorescent cells and the meanfluorescencewasdeterminedbycytofluorimetry.ThemeanfluorescencewasusedtoevaluatetheamountofR-Phycoerythrininternalizedinsidecells.

10. DeliveryofActiveCaspase-3InducesApoptosis

7

Amount of protein/cell % Fluorescent cells

HeLacellsina24-wellplateweretreatedwith15 ng of active human caspase-3 (Biovision)mixedwith5µlLipodin-Pro™ reagent. Imagesweretaken6haftertreatment(toprow).ThebottomimageisacontrolofcellstreatedwithLipodin-Pro™ reagent alone. Identical resultswereobtainedwithNIH-3T3cells.

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ells

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Caspase-3 + Pro-DeliverIN Pro-DeliverIN

µM rIN N

1 ve verI p-3

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u eli eli

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a o-D -D

Pr Pro Non

+

3 sp

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Ca

A549

HeLa

Anapoptosisassaywasperformedtoquantifyapoptoticcells.HeLacellsweretreatedwith15ngofactivehumancaspase-3mixedto5µlofLipodin-Pro™reagent in24wellplates.Aftera7-hour incubation,cellswerestainedwith both Annexin-FITC and propidium iodide. Apoptotic and dead cells were counted by cytofluorimetry. Apositivecontrolwithstaurosporine(100nM)wasusedtoinduceapoptosis.Negativecontrolswerecellstreatedwithcaspase3orLipodin-Pro™alone.

100 Inanothersetofexperiments,HeLaandA549cellsweretreatedwith15ngofactivehumancaspase-3mixedto5µlofLipodin-Pro™reagentin24wellplates.Aftera24-hourincubation,livecellswerecountedineachwellandresultsplottedaspercentagerelativetonon-treatedcells.Apositivecontrolwithstaurosporine(1µM)wasusedtoinduceapoptosis.Negativecontrolswerecellstreatedwithcaspase3orLipodin-Pro™alone.

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Stau 1 µM Caspase-3 Lipodin-Pro +Lipodin-Pro

Caspase-3 Non-treated

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Staurosporine Caspase-3 + Lipodin-Pro

Lipodin-Pro Caspase-3

Apoptotic Cells % 86 52 11 8



Overall, Lipodin-Pro™reagentcanefficientlydelivervarious typesofproteins indifferent livingcell lines.Thedeliveryefficiencyiscelltypeandproteindependentwithhigheryieldforacidicproteins.Thedeliveredproteinisstillactiveonceinsidethecellforbindingtotargetproteinsandactivatingcellularpathways.

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