Do you use PBMC or enriched T cells?

We stimulate PBMC by adding soluble anti-CD3 (33-2A3) to a final concentration of 100 ng/ml from stock 100 µg/ml.

Anti-CD28 is added to a final concentration of 0,1 µg/ml.

Best regards

Bernt

Dear Bernt,

Thank you for your reply.

I use MACS enriched T cells.

Would you please let me know in which plate you perform the assay? What was the cell number and final volume of each well?

And also would you please explain how you calculate the final concentration?(Dilution process and the volume of Ab you used)

Thank you.

Hi Maryam,

We perform ELISpot and Fluorospot in Millipore transparent 96-well plates with PVDF membranes for assay of secreted cytokines. If you just want to stimulate the cells for further analysis of the cells or the supernatant, any cell-culture plastic plate or tube would do, depending on the amount and volumes of cells that you have. 

One way to do it is to first coat the plate wells or the tube with goat-anti mouse antibodies (1 μg/ml). Then add cells at 2 × 106 cells/ml in RPMI with 10% FCS, HEPES, 2ME, penicillin/streptomycin, and glutamine with or without anti-CD3 (100 ng/ml to 1 μg/ml; good to titrate the antibody in your own system) with or without soluble anti-CD28 mAb (0,1 - 1 μg/ml). Doing it this way instead of coating anti-CD3 directly assures that the stimulating antibodies are all active.

Example: If you have a stock of antibody of 1 mg/ml (1000 µg/ml) and you want to have a final concentration of 0,5 µg/ml in your wells, your dilution is 0,5/1000, i.e. 1/2000.  So for 200 µl you should use 0,1 µl. That is inconvenient so best is to make a "working stock solution" of let's say 50 µg/ml, i.e. make a working dilution from the original antibody 1/20. Then to get to 0,5 µg/ml you add 1/100 to your 200 µl, i.e. 2 µl. 

Hope that this was of some help.

Bernt

Dear Bernt,

Thank you so much for your sincere help. I will certainly consider your tips.

Only one other question, what is the purpose of adding goat-anti mouse antibodies to each well?

Dear Maryam,

We use the same clones of anti-CD3 and anti-CD28 from eBioscience in our lab. Typically we coat round bottom 96 well tissue culture plates with 100 µL of 1 µg anti-CD3 per mL (diluted 1/1000 in PBS). We leave them in the incubator for 2-4 hours while we prepare the cells. We always use fresh PBMC or purified T cells (MACS beads, untouched Pan T cell kit).

PBMC or T cells are diluted to 1 million/mL in a suitable medium. We often use serum free medium such as X-vivo 15, because it usually gives a stronger proliferation compared to, for example RPMI with 10 % FCS and L-glutamine. Remove the anti-CD3 with a pipette tip (it's ok if there is a couple of µl left in the well), add 100 µl PBMC or T cells. Then add 50 µl anti-CD28 diluted to 8 µg/mL diluted in the same medium. We then usually add 50 µl of another reagent or medium. The final concentration of anti-CD28 in the well is now 2 µg/mL.

Kerstin

Dear Kerstin,

Would you please let me know how long you activate your cells? And what is the method which you use to see cell proliferation, and the time you can see proliferation?

(I use CFSE assay to see proliferation, but after 72h stimulation I see no difference between unstimulated and stimulated cells by flowcytometry; While under microscope I see clumps of cells. I wonder If these clumps only show cell activation, or cells really undergone proliferation too?)

Thank you so much for your help.

Dear Maryam,

Sorry about the late reply, I've been a bit busy.

I usually determine proliferation by adding tritiated thymidine the last 6 hours of a two day culture, and then read the harvested plate in a beta counter.

Sometimes I use Celltrace Violet from ThermoFisher. That works much better in our lab than CFSE. I usually have both stained and unstained cells in the culture, and CFSE seems to leak from the stained cells and then stain the unstained cells to some extent. This makes it hard to differ unstained cells from cells that have divided several times.

When I use CellTrace Violet I culture 3 to 5 days.

Kerstin

Dear Maryam,

First I have to apologize for not answering you until now. A colleague noticed that you did not get an answer on your question about goat anti- mouse antibodies.

The rationale behind using goat anti-mouse antibodies to immobilize your anti-CD3 antibodies (mouse IgG) is that you work with enriched T cells, i.e. a cell population that have fewer Fc-receptor expressing antigen-presenting cells (monocytes and B-cells). Thus, immobilized anti-CD3 work better in this situation, you get a better crosslinking of the T-cell receptor complex if you display the anti-CD3 antibodies on a surface. You can do that either by coating the anti-CD3 antibodies directly in the well or catch them on coated goat anti-mouse. In the latter case you increase the number of active anti-CD3 antibodies compared to when you coat them directly in the well.

I suggest that you test soluble anti-CD3 as well to see how it works.

Maybe you have already done these experiments during the last 9 months.

It would be interesting to know how your experiments went.

You can mail me directly at [email protected] if you want to discuss this further.

Best regards,

Bernt

Dear Bernt,

Thank you so much for your answer.

Yes, I have done my experiments fortunately. First, I tried to use plate bound anti-CD3, but it did not work in my hand. Finally, I used soluble forms of both anti-CD3 and anti-CD28 antibodies and they worked.

With best regards,

Maryam Zare

Good!

Bernt