EphA3-AP protocols

Reagents

Below is a list of  catalog numbers for reagents we have commonly used:

(product, company, catalog #)

-DMEM (without glutamine), VWR, 45000316

-L-glutamine, Biowhittaker, 17-605E

-Penicillin Streptomycin solution, Biowhittaker, 17-603e

-Trypsin/Versene (EDTA), Biowhittaker, 17-161E

-Phosphate Buffered Saline (PBS), VWR, 45000-434

-Iron-supplemented Bovine Calf Serum  (BCS), Sigma, C-8056

  (or we have also used VWR  82002-978)

-G418 sulfate, Calbiochem, 345810

              For plates, we usually use VWR cat# 25382-166. We have never really noticed any difference in quality when we have ordered media components from different companies. However, several people in our lab feel that for tissue culture plates, Falcon (BD) brand is better than Corning.

Alternative sources for these reagents:

              Recently, we have been ordering several of the above items from Invitrogen because we were able to get a discount. We haven't tested these products specifically on the EphA3-AP stable cell line, but I expect they should also work well. In case you want to try them, the Invitrogen catalog numbers are as follows:

              -DMEM, 10313039

              -L-glutamine, 25030081

              -Trypsin/Versene (EDTA), 25300054

              -Phosphate Buffered Saline (PBS), 10010064

              -Penicillin Streptomycin solution, 15070063

Optional reagents:

              For transient transfections (which I'm guessing is what you will do with the ephrinA5-AP plasmid), we use either Lipofectamine 2000, following the manufacturer's protocol, or we use the calcium phosphate transfection protocol that is attached separately. The calcium phosphate protocol is pretty easy once you have made all the solutions. In theory, the only difficulty is supposed to be in optimizing the pH of the 2xHBS - people often make solutions of pH 7.00, 7.05, and 7.10 and test to see which is best. In case you prefer to try the Lipofectamine 2000 (which is reliable, but significantly more expensive), I have included the catalog numbers of the two items you would need:

-Lipofectamine 2000, Invitrogen, 11668-019

-Opti-mem, Invitrogen, 31985-088c17

Maintaining cells

Preparation:

First, make Complete Medium (which can be stored at 4 degrees for a few months):

500 mls DMEM (without glutamine)

                 45 mls heat inactivated Bovine Calf Serum (do NOT use fetal calf Serum)

10 mls L-Glutamine solution

5 mls Penicillin Streptomycin solution

Note: It is possible to buy DMEM with glutamine already added. We buy media without and add separately because glutamine is supposed to be somewhat unstable. This way we can store the glutamine at -20 degrees for the long term. Once we thaw a bottle of glutamine, it is stored at 4 degrees, and is usually used within a few months. By the way, I think we use the glutamine at twice the final concentration some other labs do, but either way should be fine.

Also, you may notice that 45mls of BCS is not really 10% of the media. However, this is what we actually use, for practical reasons. The BCS will come in a large 500 ml bottle, frozen, and should be stored at -80 degrees. Before making your media, however, you should heat inactivate and aliquot the BCS. The procedure is as follows:

-Thaw one bottle of BCS, either in a 37 degree water bath for a few hours, or in the refrigerator overnight (or possibly over a couple of nights)

-Once the BCS is completely thawed, heat inactivate by putting in a 56 degree water bath for 30 minutes. (This step is to inactive complement components)

-Aliquot 45 mls per tube and store at -20 degrees. (If you put 50 mls of serum in a 50 ml tube, the expansion of the liquid as it freezes can break the seal of the cap.)

For Complete Media + G418, add G418 to the desired amount of media at a concentration of 500 microgram/ml. We usually dilute G418 into the media fresh for each use.

Passing cells:

Before passing cells, preheat PBS and media in 37 degree water bath.

                  -Wash cells with 10 mls PBS

                  -Remove PBS, then add 1 ml of trypsin/versene solution to 10 cm plate

              -Incubate at 37 degrees C until cells begin to detach from plate (this could take

                    anywhere from a couple minutes to perhaps 20 minutes).

-Collect cells from plate, pipetting up and down gently as needed to disperse any

  clumps

                  -Dilute cell suspension in the desired amount of Complete Media + G418, put

                    about 10 mls of media plus cells on each plate, and culture in an incubator at 37

  degrees with 5% carbon dioxide.

Note: 3T3 cells should be passed before they become completely confluent, if possible. I also would not try to split them too sparsely - no more than 1:10 from a nearly confluent plate.

Freezing cells:

Preparation:

-Grow cells until subconfluent (about 80-90% confluent) on 10 cm plates

-Make Cell Stock Media:

                  60 mls DMEM (we don't add glutamine or Pen/Strep for this)

                  30 mls bovine calf serum

                  10 mls DMSO

After mixing the above together, filter and store at 4 degrees C.

Day 1

-Pre-chill cell stock media on ice

-Wash plate with 10 mls PBS

-Remove PBS and add 1ml Trypsin/Versene; incubate at 37 degrees C until cells begin

  to detach from the plate

-Collect cells from plate, pipetting up and down gently as needed to disperse any clumps.

-Add the cell suspension to a centrifuge tube containing 10 mls Complete Medium.

-Spin down 5 minutes. We spin cells at 1000 to 1200 rpm in our tabletop Beckman

  centrifuge, which if I calculated correctly, should be somewhere in the range of 200 g.

-Remove sup and resuspend cells with 1 to 3 mls ice-cold Cell Stock Media

-Transfer 1ml cell suspension into each vial (for example, VWR cat# 66021-972)

-Put vial(s) in -20 degree freezer as quickly as possible (keep on ice until you move

   it to the freezer)

-After 2 hours at -20 degrees, transfer vial to -70 degree freezer and keep there overnight.

Day 2

-Transfer vial to liquid nitrogen tank

Thawing cells:

-Pre-warm Complete Medium +G418 at 37 degrees

-Remove tube from liquid nitrogen and thaw in 37 degree water bath.

-Immediately after thawing, transfer cell suspension drop by drop into a centrifuge tube

  containing 10 mls of Complete Medium +G418.

-Spin down 5 minutes.

-Remove sup and resuspend in 10 mls Complete Medium +G418. Transfer cell suspension to a 10 cm  plate, and culture as usual (37 degrees C, with 5% carbon dioxide).

Additional comments:

I will be sending 2 vials of the EphA3-AP (Mek4-AP) cell line. When you receive them, store one in liquid nitrogen (or at -80, if that is not possible) for back-up, and thaw the other one directly. When this plate is nearly confluent, split cells into 5 plates. When these plates become subconfluent, freeze down cells from 4 of the plates, and split as desired to maintain the cells from the other plate.

If for any reason you want collect EphA3-AP sup that does not contain G418, culture the cells until they are confluent, wash and replace with Complete Media (without G418) and culture for several days (4-6, or until they nearly die), then collect, filter, and store sup at 4 degrees as usual.

For transient transfections, we usually use 293T cells (though I understand COS are also supposed to work fairly well), and we handle these cells in basically the same way, except that G418 is not included in their media.