Hi everyone,
Been trying to culture murine T cells isolated from spleen or lymph nodes (iLNs) but been having difficulty in keeping the Pan T cells alive even after only 24hr of culturing (viability drops to ~20%). I process the tissue after harvesting with a plunger and 100um cell strainer in cold cRPMI to ensure single cell suspension, wash with PBS/2% FBS and performed RBC lysis with 3ml ACK lysis for 2 mins, quench with complete RPMI (cRPMI). Then isolate Pan T cells using the Myltenyi Pan T cell isolation kit (cat no:130-095-130 and stimulate with Myltenyi anti CD3 / or anti-CD3/CD28 beads or keep them resting for 48hr iin the 96-well plate at 0.25x10^6/well.
** Everything takes place at 4 C with cRPMI (10% FBS, 2mM Glutamine and 1mM sodium pyruvate, 100 units penicillin and 100μg/mL streptomycin and 1x NEA). For culturing i added to the cRPMI media: 0.05mM 2-Mercaptoethanol (2ME).
I have trouble-shouted the following and have NOT made a difference:
1) processing in cold cRPMI vs PBS or PBS/2%FBS (cRPMI elevated the viability by 20% vs PBS)
2) 2ME concentration: titrated between 50mM to 0.05mM and it seems that 0.05mM is the same with not adding 2ME (which was only around 20%) and anything above that concentration decreased the viability even more.
3) PBS tablets versus premade PBS from Thermo (pH checked)
4) stimulation induced cell death (although you always see some but even the resting cells die )
5) ACK lysis (optimised time required for lysis and quenching and volume) and also compared T-cells that went through RBC lysis (Splenocytes) vs cells not undergone through RBC (LN). Findings: had equally bad viability.
6) compared resuspending the cells with mechanical mixing (racking the tube with cells on a rack to release the pellet and then resuspending with the pippete) vs resuspending the pelleted cells with pippete directly . Findings: still did not make a difference
7) Centrifuge speed 1500rpm vs ~1200rpm (300xg). Did not make a difference.
Thank you all in advance, any help would greatly be appreciated.
Hey Charys,
Maybe you can add some IL-2 to the medium? We recommend to add 50U/ml IL-2 in our T cell expansion protocol (see attachment).
Did you compare sorted T cells vs. seeding whole splenocytes? From my experience, the T cells are happier when you expand them in whole splenocytes (after a few days of expansion, you have around 90% T cells anyway).
I usually sorted CD4+ T cells and they didn't like it when the cell density was too low. So maybe you can also try to increase the cell density?
Best,
Alex
I agree with Alexandra Nordlohne . Add IL-2 in cultures.
Also, what plates are you using for culturing? I would suggest use TC treated 96 well plates and since you are using dyna-beads, use U bottom plate instead of flat bottom.
Moreover, I would suggest you to try TC treated 96 well flat bottom plate, coat with CD3 (5ug/ml) overnight and then culture your cells in media+IL-2 along with soluble CD28 (2ug/ml) with a seeding density of 0.125X10^6 cells/well in 200ul media.
Good luck!