Cultivation of Human Embryonic Kidney 293 Cells

Medium:

500 mL Dulbecco's Modified Eagle (DME-HG)
50 mL Fetal Bovine Serum (FBS) - 10 % of the medium volume
5 mL penicillin/streptomycin (10 000U/mL Pn and 10 mg/mL Str)-1x final concentration
Trypsin (0.05% solution with 0.5 mM EDTA)
Phosphate Saline Buffer (PBS), pH 7.0-7.2

Procedures:

Starting from a Cryotube:
Prewarm the medium at 37°C and fill one 15 cm dish with 25 mL. DO NOT USE MEDIA THAT CONTAINS HYGRO. , G418, etc. AT THIS POINT!
Rapidly thaw the cells in your hand, gently pippet up and down to thaw any remaining ice before distributing in a dish filled with warm media.
Rock the dish to distribute the cells. Do not swirl-if you do they will all clump towards the center of the plate.
Change the medium after 24 hrs or once the cells have attached.
Splitting Cells (all solutions must be prewarmed):
Prepare a new dish with fresh medium. (25 mL for 15 cm plates, 10 mL for 10 cm plates)
Aspirate the old medium from the dish.
Wash the cells carefully with PBS to remove residual medium and FBS which can inhibit the trypsin.
Trypsinize cells by adding 3-5 mL of trypsin to the dish and incubate at 37°C (or RT, it depends on its activity) until cells have detached (usually 2-5 minutes).
Take a fraction of the cell solution and transfer it to the new dish (BSA in medium inhibits the trypsin).
When splitting confluent 293 cells in a 1:10 ratio, confluence is reached again after 3-5 days.

Cultivation of HEK 293 in Roller Bottles

Medium:

500 mL Dulbecco's Modified Eagle (DME-HG)
50 mL Fetal Bovine Serum (FBS) - 10 % of the medium volume
5 mL penicillin/streptomycin (10 000U/mL Pn and 10 mg/mL Str)-1x final concentration
Trypsin (0.05% solution with 0.5 mM EDTA)
Phosphate Saline Buffer (PBS), pH 7.0-7.2
***The roller bottles used are ribbed, with ~ 1700-2200 cm2 of surface area.
If you want to use bottles with 2200 or more cm2 surface area-you must increase the amount of cells you innoculate each bottle with.
This is very important if you wish to avoid cell patches that will degrade the quality of your culture.

Procedures:

Aspirate the media (it might also be harvested in order to collect your protein).
Pour directly in each roller bottle 350 mL of prewarmed media.
Place the roller bottles in the incubator and let them roll at 0.1 rpm to warm up.
Wash each 15 cm plate with approximately 10 mL PBS.
Trypsinize the cells with 5 mL trypsin ( do not trypsinize more than 5 plates at a time).
Pipette the suspension from ONE plate into ONE roller bottle.
Put the roller bottles in the incubator and let sit overnight at 0.1 rpm to allow the cells to attach.
When the cells are attached onto the bottles' surface (After ~ approximately 24h), increase the rotation to ~1rpm.
Change the media once the cells reach confluence.
At this point we usually change into serum free media (if that's what is called for), or just add fresh media and continue the culture.
***If using serum free media-after the first harvest (or once the cells reach confluence) wash each bottle once with ~50 mL serum-free media.
After washing, add back app. 250-300 mL of media lacking serum. Harvest the serum free media after 2 days. Repeat if the cells appear healthy.

Transfection of HEK 293 Cells

Reagents:

Lipofectamine (LFA) - Gibco, cat. # 18324-012
Opti-MEM (OM) - Gibco, cat. # 31985-070
DME-HG/ FBS, which contains:
500 mL Dulbecco's Modified Eagle (DME-HG)
50 mL Fetal Bovine Serum (FBS) - 10 %
and
2) DME-HG/ FBS/ Pn/ Str, which contains:
500 mL Dulbecco's Modified Eagle (DME-HG)
50 mL Fetal Bovine Serum (FBS) - 10 %
5 mL penicillin/streptomycin (10 kU/mL Pn and 10 mg/mL Str)
Phosphate Saline Buffer (PBS), pH 7.0-7.2
Trypsin (0.05% solution with 0.5 mM EDTA)

Procedures:

Split HEK 293 cells, grown in DME-HG/ FBS/ Pn/ Str into 6-well uncoated or Poly-D-lysine coated plate with 2-3 mL medium.
You can get ~18 wells (~90% confluent the following day) from a 15 cm confluent dish.
When cells reach 70-90% confluence or so, wash each well with 2 mL PBS and change the media to antibiotic free DME-HG/ FBS.
Antibiotics are significantly more toxic to cells after treatment with Lipofectamine.
Dilute 8 uL Lipofectamine into 150 uL prewarmed (37°C) Opti-MEM.
Dilute 2.5 ug plasmid DNA, containing your gene of interest, into 150 uL Opti-MEM.
Incubate 5 min. at room temperature.
Mix both DNA/Opti-MEM and LFA/Opti-MEM solutions and let them sit 20 minutes at RT.
Add nucleolipid complexes directly onto the cells and incubate for 24 h at 37°C.
Change the media after 24h (carefully aspirate the old and pipette new) with DME-HG/ FBS/ Pn/ Str and incubate for an additional 24 h.
If the cells are too confluent-you may split the cells immediately into 15 cm dishes. You must, however, wait an additional 24 h before adding hygro.
Split the cells into 15 cm plates with DME-HG/ FBS/ Pn/ Str, containing 150 ug/mL Hygromycin B or 400-500ug/mL G418.
Incubate until foci appear (appr. 8-9 days). Change media every 3-5 days.
When most of the "non-stable" cells start dying as a result of the drug selection (app. on day 5) the media must be changed.

Transfection of 293 cells in 10 cm dishes
******Abbreviated protocol******
Surface area of 10 cm dish is 8 x greater than that of a 6-well dish.

Therefore:
1. Use 20 ug of plasmid DNA.
2. Use 64 uL of Lipofectamine 2000 per dish
3. Place each into 1.2 mL of Opti-MEM.
4. Let sit at RT for 5 minutes.
5. Mix and let stand 20 minutes prior to addition to cells.
6. Cells should be in ~8 mL of media (w/o drugs of any kind).
Continue as usual.

Transfection of 293 cells in 15 cm dishes
******Abbreviated protocol******
Surface area of 15 cm dish is 18 x greater than that of a 6-well dish.

Therefore:
1. Use 45 ug of plasmid DNA.
2. Use 144 uL of Lipofectamine 2000.
3. Place each into 2.7 mL of Opti-MEM.
4. Let sit at RT for 5 minutes.
5. Mix and let stand 20 minutes prior to addition to cells.
6. Cells should be in ~20 mL of media (w/o drugs of any kind).
Continue as usual.

Checking for Protein Expression

The following protocol is useful for analysis of secreted proteins which have C-terminal Fc-tag.

Reagents:

Protein A-Sepharose (Amersham cat. # 17-1279-01)
HBS buffer
HBST buffer
Procedures:
*NB For each sample (1.5 ml) use 50 uL Protein A-Sepharose.

Prewash sepharose once with equal volume dd H2O (H2O removes ethanol in which the Sepharose has been stored).
Spin down for 1 min at max speed in microcentrifuge (14 000 rpm) and carefully aspirate the supernatant.
Wash with the same volume HBST. Centrifuge for 1 min at max speed.
Carefully aspirate the supernatant (repeat washing twice).
Resuspend Protein A-Sepharose in an equal volume HBST. For example, if you have 150 ul beads-add 150 ul HBST.
Take 1.5 mL of media, place in a microfuge tube, and mix with 50 uL Protein A-Sepharose in HBST.
*Protein A Sepharose should be transferred to the tube with a cut pipette tip which prevents the sepharose beads being damaged.
Put the tubes on a rocker for 2 h at 4 degrees. The longer the incubation, the better.
Centrifuge for 1 min at max speed in microcentrifuge (14 000 rpm) and aspirate the media.
Add 1 mL HBST and rock the tubes gently until the beads get mixed with the solution.
Centrifuge for 1 min at max speed (14 000 rpm) and aspirate the supernatant.
Rinse with 1 mL HBS, spin it again and aspirate the supernatant.
Add 30 uL 1x SDS-PAGE sample buffer to the beads.
Boil for 3 min at 95 degrees. Flick the tube couple of times while boiling so the beads get mixed well with the sample buffer and the protein gets extracted from them to the solution.
Spin down for a minute at 14 000 rpm.
Carefully remove supernatant with loading tip and load onto an already prepared SDS-PAGE gel (% depends of the MW of the protein).

A background band at ~60kDa and possibly residual BSA at 67kDa will be present-so keep this in mind if your protein is about this size.

Western Blot

If your construct (target gene) does not contain any tags an antibody against your favorite protein might be used.

Materials:

Antibody (AB) against target protein, which has been bound with chromophore, enzyme and etc., to produce specific signal.
Whatman 3M chromatography paper.
PVDF or nitrocellulose transfer membrane.
Blocking agent (e. g. fat free milk powder, 4% solution, prepared before using).
Western transfer buffer
TBST buffer
Procedure:

*For this procedure the protein expression should be checked in medium without FBS so that BSA in the serum does not interfere.

Mix 30 uL of media with 10 uL 4x Sample buffer. Boil the samples for 5 min at 95 C.
Load onto a SDS-PAGE gel and run for ~1 h at 200V.
Transfer.....
Remove membrance from transfer unit and rinse the membrane with TBST.
Block the membrane with 15 mL 4% fat free milk ( dissolve 2g of fat free milk in 50 mL of TBST). Incubate for 1h.
Rinse the membrane briefly with TBST.
Incubate the membrane with the primary antibody against the protein (typically 1:1000 diluted in TBST) for 2h or overnight (preferable) on a rocker at 4 C.
A total volume of 15 mL of the antibody solutions is sufficient to cover the membrane.
After the incubation, rinse the membrane 3 times for five to ten minutes each with TBST.
If the primary antibody is not directly conjugated to the signal providing molecule(enzyme, chromophore etc.), a secondary antibody against the primary is needed.
Dilute the secondary antibody in TBST according to the company instructions and incubate for ~ one hour.
After the incubation, rinse the membrane 3 times for five to ten minutes each with TBST.
Develop the signal by either adding an appropriate colored substrate for the enzyme or by measuring the induced light for the chromophore.

Storage of Transfected HEK 293

Cells should be stored by freezing at -80°C( up to 3 months) or in liquid nitrogen(~-196°C) for several years.

Reagents:

Media:
DME-HG/ FBS/ Pn/ Str which contains:
500 mL Dulbecco's Modified Eagle (DME-HG)
50 mL Fetal Bovine Serum (FBS) - 10 %
5 mL penicillin/streptomycin (10 kU/mL Pn and 10 mg/mL Str)
DME-HG/ FBS/ Pn/ Str/ containing 10% DMSO (kryoprotector)
Trypsin (0.05% solution with 0.5 mM EDTA)
Phosphate Saline Buffer (PBS), pH 7.0-7.2

*The amount of time the cells spend in trypsin should be as short as possible. Keep this in mind when going through this procedure.

Procedures:

Aspirate the media.
Wash the plates with ~10 mL PBS.
Trypsinize the cells with 3 mL trypsin.
Put the trypsinized cells in a 15 mL Falcon tube with 7-10 mL DME-HG/ FBS/ Pn/ Str (the trypsin is inhibited by the serum).
Centrifuge them for 5 min at 600 RPM (~100-200 x g).
Aspirate as much of the supernatant as possible.
Resuspend the cell pellet gently, but thoroughly, in 3 mL DME-HG/ FBS/ Pn/ Str/ 10% DMSO.
Aliquot the suspension in freezing vials placed in a styrofoam box (app. 1 mL in each vial) and place immediately in the freezer (-80°C).
The styrofoam will sufficiently slow the cooling of the cells.
After 24h the vials should be moved to liquid nitrogen.

Prepare media for labeling:

1. Add 30 mg/L L-cystine-2HCl to DME-HG lacking Met. & Cys. (Gibco 21013-024).
2. Add 60 mg/L L-selenomethionine.
3. Let sit overnight at room temperature to allow the cys. & se-met. to go into solution.
4. Filter sterilize and add Pen./Strep. and L-glutamine to 2mM (Do not add glutamine prior to this step!)

Label the cells:
1. To cells that are ~90% confluent, aspirate away media, and wash with media W/O Met. and Cys. (preferred).
2. Add Se-Met media to the cells and let incubate for 12 hours.
3. Remove the media and feed with fresh media. Throw this media out. This step serves to deplete intracellular methionine pools.
4. Continue to incubate for 48-72 hours.


**Cells labeled in this way have ~92-93% incorporation of seleno-methionine.