Methods with mammalian cells

•  Cell culture of 293, HeLa or MEF

•  Freezing of Mammalian Cell Lines

•  Thawing of Mammalian Cell Lines

•  Transfections

•  CaCl₂ Transfection

•  Polyethylene-imine Transfection

•  Lipofectamine Transfection

•  Electroporation of mammalian cells

•  Electroporation of Endothelial Cells.

•  Lentiviral Transduction.

•  Generation of Stable 293 Cells.

•  Reporter Gene Assays.

•  Luciferase Assay.

•  β-Galactosidase Assay with CPRG.

•  Cell Viability Assays.

•  Crystal violet staining (as proliferation or cytotoxicity assay)

Cell culture of 293, HeLa, or MEF Cells

Solutions:

Dulbecco’s modified Eagle’s medium complete (DMEM complete) containing 10 % FCS (fetal calf serum), penicillin (100 u/mL), streptomycin (100 µg/mL), glutamine 2 mM (stock solutions of penicillin, streptomycin and glutamine: 100x)

DMEM G418 medium: DMEM complete containing G418 500 µg/mL

G418 stock solution: 500 mg/mL G418 in Hepes 100 mM

PBS deficient: NaCl 8 g, Na₂HPO₄ 1.44 g, KH₂PO₄ 0.24 g, with AD to 1 L, pH adjustment with HCl to pH 7.4 (provided by Novartis facility)

Trypsin solution 0.25 % in Hepes 10 mM

293 cells (immortalized human embryonic kidney cells, adherent), HeLa (human cervix carcinoma cells, adherent) and MEF (mouse embryonic fibroblasts, adherent) are cultured in DMEM complete medium, in addition to this DMEM complete medium, the medium for stable 293 cell lines with a genomic integration of a neomycin-resistance plasmid containing the gene of interest, contains 500 µg/mL G418 as permanent selection agent. Cells are cultivated in 75 cm² tissue culture flasks at 37°C, in a humidified (90 %) and CO₂ (5 %) containing atmosphere. All cell types are trypsinized and passaged every second or third day in proportion of 1:4 or 1:6, respectively. For passaging the cell culture medium is removed, about 13 mL of PBS def. (room temperature) are used to wash the cells. PBS def. is removed as well and about 3 mL trypsin solution (37°C) are pipetted into the flask and spread evenly all over the bottom. Excess of trypsin is removed and the flask is placed in the incubator for around 3 minutes. With an inverse microscope (Nikon TMS F 4x or 10x objective) it is checked if the cells are already detached and round in shape. Trypsinized cells are gently resuspended by several times up/down pipetting in 8 mL (to 12 ml – according to the desired passaging ratio) complete DMEM and 2 mL are transferred into a new tissue culture flask containing 13 mL of DMEM complete or DMEM complete including G418. Tissue culture flasks prepared for MEFs have to be gelatinized prior to passaging as follows: 15 mL of 1 % gelatin solution (autoclaved and sterile filtered, 37°C) are pipetted in a 75 cm² tissue culture flask and incubated at 37°C for 30 minutes, the gelatin solution is discarded afterwards. After this procedure, passaging is performed as usual.

Freezing of Mammalian Cells

Solutions:

Freeze medium: DMEM, 10 % (v/v) DMSO (dimethylsulfoxide), 20 % (v/v) FBS (fetal bovine serum), sterile filtered

293, HeLa, MEF, stable transfected 293 cell lines are passaged as described above, seeded in 75 cm² cell culture flasks and grown until 100 % confluency is reached. The cells are trypsinized as described above, resuspended in DMEM complete medium (4°) and centrifuged at 1000 rpm (400 g), 4°C for 5 minutes in a cell centrifuge with a swing-out rotor. The supernatant is removed and the cell pellet of one 75 cm² cell culture flask is resuspended in 2 mL freeze medium (4°C). 1 mL of resuspended cells are pipetted in pre-chilled cryotubes, placed in an isopropanol-box (“Mr Freeze”, 4°C) and then transferred to -80°C in order to freeze the cells in a controlled and slow procedure. After 24 hours the cell aliquots are transferred and stored in the liquid nitrogen tank at -196°C.

Thawing of Mammalian Cells

A cell aliquot of 293, HeLa, MEF or stable transfected 293 cell lines stored in liquid nitrogen is quickly thawed in a 37°C water bath with constant moving to prevent local overheated spots warmer than 4°C. The thawed cell suspension is resuspended and diluted in about 20 mL DMEM complete medium and the cells are centrifuged at 1000 rpm for 5 minutes at 4°C. The supernatant is removed by suction, the cell pellet is resuspended in DMEM complete medium or supplemented with G418 and the cells are seeded in 75 cm² cell culture flasks.

Transfections

CaCl₂ Transfection

Solutions:

CaCl₂ 2 M in Aqua dest:

HeBS (Hepes buffered saline): Hepes 50 mM, NaCl 280 mM, Na₂HPO₄ 1.5 mM, pH adjustment to exactly pH 7.05 with HCl, sterile filtered (0.2 µm pore size), stored at -20°C

Preparation: 293 or HeLa cells are passaged and seeded at a density of 500.000 cells/6 well (40 % confluence). After one day (80 % confluence) transfection is carried out. Transformation mix: 2 µg of DNA (1 µg/µL) are diluted in 60 µL of aqua dest. in a  polystyrene tube and 9 µL of 2 M CaCl₂ are added and mixed, 71 µL of HeBS are added dropwise by unscrewing a P200 Gilson Pipette while constantly vortexing at half speed. After incubation of 5 minutes at room temperature the solution is pipetted dropwise as evenly as possible on the surface of the cell culture dish. Once the DNA/calcium-precipitates get in contact with the cells the tissue culture dish should not be moved a lot. Normally the proteins of interest are expressed sufficiently after 24 hours of transfection (when CMV promoter containing plasmids are used).

Polyethylene-imine Transfection Method (for 293 and HeLa)

1.    Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE: Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 2006, 138:85-98.

Linear PEI (0.323 g/L in A. dest = 7.5 mM): stirred on low heat for 1 h until all particles are in solution, pH set to 7.0

PROTOCOL for PEI transfection (Kalsoom)

Mix the contents of soln A and B separately

Add sol B to sol A, mix and vortex briefly.

Let it at room tepmrature  for 15-20 minutes

Wash the cells with SFM once and then add SFM:

Drop wise add the transfection mix to the wells

Put in the incubator at37C.

Change the SFM with full media after 3-4 hours

Incubate for 24-48 hour at 37C:

Solution: PEI 25000 sigma  density 1.03g/ml

Stock soln. of PEI 40mM: 10g PEI in 10 ml distilled water Mix 4-5 hours,

Working soln. 87ul of this in 100ml water. Mix 4-5 hours maintains pH 7.0 with HCl. Filter steril 2times and store at 4C.

2X HEPS pH 7.05: (500ml): 8gNaCl, 0.2g Na2HPO4.7H2O, 6.5g Heps or 5.96g Reeacid. Filter sterile and store at -20C.

Lipofectamine Transfection

Solutions:

Sterile DMEM medium without any additives (DMEM Æ)

DMEM complete

Lipofectamine Plus reagent

Lipofectamine reagent

Preparation: 293 or HeLa cells are passaged and seeded at a density of 500,000 cells/6 well (40 % confluence). After one day (80 % confluence) transfection is carried out. For transformation of one 6-well 293 (HeLa) cells, 100 µL DMEM Æ medium is mixed with 1 µg (1.5 µg) plasmid DNA and 4 µL (5 µL) lipofectamine Plus reagent and incubated for 15 minutes at room temperature. 100 µL of DMEM Æ are mixed with 3 µL (3 µL) lipofectamine reagent added to the incubated solution and mixed gently. The lipofectamine/DNA solution is further incubated at room temperature for 15 min. Thereafter, the transformation mix is diluted with 800 µL DMEM Æ. The culture medium of 293 or HeLa cells is removed by suction, the cells are carefully washed once with at least 1.5 mL DMEM Æ medium, the wash DMEM is removed and the transformation mix is carefully added to the cells. The 293 (or HeLa) cells are incubated with the transformation mix in the incubator for 3 hours (7 hours). After the transformation period the transformation medium is removed and replaced by DMEM complete medium. 1 day after transfection, cells are investigated.

For Lipofectamine2000 transfection follow the guidelines of the manufacturer (transfection is possible in presence of serum)

Electroporation of mammalian cells

Electroporation is the cost effective method for introducing foreign DNA into the primary and other hard to transfect cells. In this protocol HUVEC transfection is shown as an example. Electroporation was carried out when the cells were at passage 5.

1. One day before electroporation, the cells were trypsinized and seeded in 100cm cell culture flasks.

2. The next day the cells were ready for electroporation, the cells were washed with sterile PBS, harvested by trypsinization and centrifuged at 4°C.

3. The cells were resuspended in electroporation buffer, counted and 5.4x106 cells/400ul was used for each transfection. Every step was carried out at ice.

4. 20 µg of the plasmid DNA was used for each electroporation. The DNA was mixed with cells and transferred to 4mm electroporation cuvette and placed on ice.

5. The cells were than electroporated at 960 µF and 200volts and put immediately on ice.

6. The electroporated cells were mixed with 6ml of media and seeded in 1% gelatin coated dishes. The cells from one electroporation are enough to put in three wells of a 6-well plate.

7. After 4 hr the media was changed. The cells were analysed after 24-48 hr of transfection.

Electroporation Buffer:

       20mM HEPES

       135mM KCl

       2mM MgCl2

       0.5% Ficoll 400

       pH 7.6 with NaOH

TIPS:

1. DNA  eluted with water works better than TE buffer.

2. Proceed quickly after mixing DNA and cells. Keeping DNA and cells mixture longer results in low efficiency.

3. After electroporation , before seeding the cells place them on room temperature for 10 min.

Electroporation of Endothelial Cells

(Angiogenesis 2004; 7:235-41)

day one:

seed cells for electroporation: 5,4 * 10⁶ / T162

day two

à prepare new  culture dishes (+medium and fibronectin) before starting electroporation

       and place in incubator in the meantime

à prepare eppis with DNA, prepare + label electroporation cuvettes

ECs should be 70-90% confluent

wash cells with HBSS^def once, harvest cells by trypsinization

stop with RPMI 1640 (Gibco) + 10% FCS (4 °C)

centrifuge 5 min at 4 °C, 1300 rpm

resuspend ECs in 1 ml RPMI 1640 (Gibco)  + 10% FCS

count cells (in the meantime: put cells and medium on ice)

dilute ECs to 2,5 – 5*10⁶ cells/ml in RPMI 1640 + 10% FCS (4 °C)

mix 20 µl plasmid DNA with 400 µl cells, incubate eppi on ice for 10 min

transfer mix to 4mm-cuvette (BioRad electroporation cuvette) and electroporate immediately at 200 V, 1200 µF (should result in 40-45 msec pulse length)

Note: alternatively, 960 µF are also acceptable (pulse length: 30-35 msec)

leave cuvette at RT for 10 min, then re-seed cells in prepared 10cm-culture dishes with 10 ml EGM2-MV and fibronectin

analyse cells after 24-48 h  

Expected transfection efficiency:    50-70 %

Expected cell death:    20-50 %

Lentiviral Transduction

Day1:

4x106 293 cells were plated in 10cm2 tissue culture plate in 10ml of DMEM, 24 hours before transfection.

Day2:

At the time of transfection, the plate was taken out of incubator, 5ml of media was sucked out from the 10cm2 plate and chloroquin was added to the final concentration of 25uM. The plate was put back in incubator and DNA was mixed in a falcon tube in the following order:

i.          5ug of VSVG (envelop protein)

ii.         7.5ug of packaging protein

iii.         10ug of the Target vector

After mixing the DNA,125ul of 2M CaCl2 and 875ul of H2O were added by tapping gently.

1ml of 2xHBS was then added drop wise into the DNA while bubbling with a pipette. When finished the mixture was continued to bubble for 1 min. The plate was taken out of the incubator and transfection mixture was added drop wise on the plate. The plate was swirled gently and returned to the incubator. After 4 hours of transfection the media was replaced with the fresh DMEM and the plates were transferred to the virus room incubator.

Day4:

48 hours later the viral supernatant was harvested and filtered through 0.45um filter. To the filtrate Polybrene was added to the final concentration of 10ug/ml and the filtrate was added to the target cells for 24 hours.

Day5:

After 24 hours the filtrate was removed, cells were washed with PBS and the fresh medium with the antibiotic (puromycin) in the final concentration of 1ug/ml was added to the cells. The cells were grown in the resistant media until all the cells in the control (untransfected) flask were dead. The cells were then checked for transfection and proceeded for the desired assay.

Generation of Stable 293 Cells

293 cells are seeded at a density of 500,000 cells/6-well. The next day CaCl₂ transfection with linearized and EtOH/acetate precipitated plasmid DNA is carried out as described above. 1 day after transfection cells are trypsinized and reseeded in a 10 cm cell culture Petri dish (about 60 cm²) and DMEM complete medium is exchanged with DMEM complete medium containing 900 µg/mL G418. About 2 weeks later, colonies that survived the G418 selection are checked by fluorescence microscopy for the expression of the transfected GFP-fusion protein and positive colonies are picked under the microscope (4x magnification objective) under sterile laminar flow by scraping them off with the tip of a pipette, careful suction of the colony and placement into a well of a 24-well plate containing the selection medium. 1 d after the isolation, the adherent colony is separated into single cells by trypsinization and reseeding of the cell suspension in the same well. After reaching confluence, the cells are further expanded up to 75 cm² flasks and aliquots are frozen in liquid nitrogen.

Reporter Gene Assays

Luciferase Assay

(Naila Malkani)

In the firefly luciferase activity assay D-luciferin is used as substrate for firefly luciferase. The oxidation of luciferin to oxyluciferin in the presence of ATP and magnesium is catalysed by luciferase, which results in bioluminescense.

Day1:

Seed the cells in 24 well plate, according to the experiment design.

Day2:

Transfect the cells with reporter plasmid, beta-gal and gene of interest with proper positive and negative controls.

Day3:

After 26-30 hrs of transfection lyse the cells. Take out the media, wash the cells with 1xPBS and put 50μl of lysis buffer (PI added). Transfer the plate to - 80°C.

When you want to proceed with the assay, thaw the plates and transfer the contents of each well into the eppendorf tubes. Centrifuge at 14000rpm at 4°C for 20 minutes. Use the supernatant for proceeding the assay.

In a 96well white plate add 20μl of the supernatant and 50μl of the assay buffer. Measure the luminescence at the reader (synergy) by injecting 50μl of the injection buffer.

For normalization, measure beta-gal activity. Transfer the contents of the above plate (almost 110μl from each well) to a normal transparent 96 well plate and 50µl of beta-galactosidase substrate (CPRG) was added to each well. The yellow CPRG solution turned orange to red depending on the beta-galactosidase activity which is measured at 595nm. The normalized activity is measured by dividing firefly luciferase values to respective beta-galactosidase values.

Lysis buffer:

0.1M KH2PO4 (pH 7.8  by NaOH) 

Assay buffer: (1ml)

MgSO4 (1M)                           =      20ul

ATP (100mM)                         =    200ul

Glycyl gliaia buffer (25mM)    =    780ul

Injection buffer: (3ml)

1M Luciferin                                   =    750ul

Glycyl gliaia buffer 25mM              =   2250ul

Recipes:

0.1M KH2PO4

136g KH2PO4 / 1 liter mQ H2O pH 7.8  by NaOH .

25mM Glycyl glycine

3.3g of glycyl glycine /1 liter mQ H2O pH 7.8

1mM luciferin

14mg D-Luciferin +3.5ml TrisHCl pH 7.5  make volume upto  50ml with mQ H2O.

CPRG

27.1mg of CPRG in 20ml PBS+0.5%BSA

TIPS:

After getting supernatant, use immediately for assay, avoid repeated freeze thaw.

Warm the luciferin to 37°C before using.

β-Galactosidase Assay with CPRG

(Johannes Schmid)

Lysis Buffer:

0.25M Tris/HCl pH 7.4 (or better 8.0)

0.25% (v/v) NP40

2.5 mM EDTA 

CPRG-substrate solution: 1 mg/ml (= 1.65 mM)

in PBS + 10 mM KCl, + 1 mM MgCl₂

alternative substrate buffer:

60 mM Na₂HPO₄ pH 8.0, 1 mM MgCl₂, 10 mM KCl, 50 mM Mercapto-ethanol

Stop solution: 0.5M Na­₂CO₃

Lyse cells with lysis buffer.

Pipet about 10 µl of extract into a 96-well plate (appropriate neg. control / blank: 10 µl of mock transfected cells, or non-transfected cells – as there is a slight endogenous b-Gal activity) – leave one well empty for blank (A-1)

Add 100 µl substrate solution to the wells (also to the blank-well)

Incubate until red color develops (min to hours – depending on b-Gal activity, if you have low activity you can also incubate at 37°C)

Optional: Stop with 50 µl of Stop solution (only necessary if you want to time it exactly, e.g. by adding the substrate in a timed way and stopping the reaction in the same way)

Measure with  ELISA Reader at 570 nm (Filter #3)

Cell Viability Assays

Crystal violet staining

(can be used as proliferation or cytotoxicity assay)

HeLa cells and transfectants derived thereof were plated at a density of

1.5 x 10⁴ cells/well in triplicates in 96-well microtiter plates in 100 µl of

Click RPMI 1640 overnight at 37°C. On the next day, the reagents of

interest were added in the presence of 2.5 µg/ml cycloheximide. The plates

were incubated for additional 12- to 24-h culture, and cell viability was

determined by crystal violet staining. Briefly, supernatants were discarded

and the cells were washed once with PBS, followed by crystal violet staining

(20% methanol, 0.5% crystal violet) for 15 min. The wells were washed

with H2O and air dried. Residual dye was diluted with methanol for 15 min,

and OD at 550 nm was measured with a R5000 ELISA plate reader (Dynatech,

Guernsey, U.K.).

Ref.: The Journal of Immunology, 1998, 161: 3136–3142.

Example: