Hello,
I need to isolate splenocytes from pig spleens and I have some doubts about the protocol that I hope some can clarify me:
1. Red blood lysis buffer --> I have read that the splenocytes can be sensitive to the RBC lysis buffers and a long incubation time with this types of buffers could damage the cells. What kind of RBC buffer and for how long I should incubate the cells in it? Also, is better if I incubate the cells on ice or RT?
-Time and speed of centrifugations --> The protocol that I got from mouse splenocytes isolation, indicates centrifugations of 10 minutes at 400g. I was wondering whether I should use the same speed for the pig splenocytes isolation.
-ILs used for the Lymphocytes activation --> I would to enhance the proliferation of Lymphocytes T after the splonocytes isolation. If I am not wrong, I should add IL2 and IL12 to the media (RPMI) for that purpose, is that correct? Could someone advise me about the ILs that I can use to the media to enhances Lymphocytes T proliferation?
Thank you very much in advance for your help.
Best wishes
Iker
Hi Iker, I have no experience with pig splenocytes so I can only offer my insights from mouse splenocytes.
1. RBC Lysis Buffer: We regularly use ACK (Ammonium Chloride) RBC lysis buffer
NH4Cl 4.15 g
KHCO3 0.5 g
EDTA 0.0186 g
Make up to 500 mL in MQ water
pH 7.2-7.4
Filter sterilise
Some people think you are better to leave the RBCs alone as there is the risk of some damage to the lymphocytes, plus you are eliminating the potential impact of the RBCs (which are there in vivo), and they tend to just die off in culture anyway. Having said that, it does make counting a bit easier when their numbers are seriously reduced. We use 2 mL warm ACK buffer per mouse spleen, incubate for 3 mins at room temp, then dilute with at least 3 X DPBS. A pig spleen is so much bigger so you'll have to play around with scaling up those volumes.
2. Time & speed of centrifugations: We use 300 x g for 5 mins in a 15 mL Falcon Tube or 7 mins in a 50 mL Falcon tube. Splenocytes are quite small so they should be OK at anywhere between 300-400g
3. ILs for Lymphocyte activation: Different cytokines do different jobs, some promoting survival, others promoting proliferation. I would recommend Suzanne Heinzels work on T cell proliferation. She has some excellent data on which cytokines to use to promote T cell survival and/or proliferation. If you wish to stimulate non-specific proliferation anti-CD3 and/or anti-CD28/CD27 mABs will do the trick. I would highly recommend that you do dose titrations of all cytokines that you use to make sure that what others have published works for you. You can't simply take what's used for human work and apply it to mouse cells. The same will most likely apply to your pig cells.
All the best
Hi Melanie,
Thank you very much for your answer, I really appreciate it. All the information you said is really useful for me so I am really grateful.
I will read Suzanne Heinzels work as you recommend me to do.
Kind regards,
Iker
You're welcome Iker. I apologise, I wrote 3 x dilution of DPBS for the RBC lysis buffer, that should have read at least 10 X dilution :) So I add 2 mL of RBC LB & then top it up to around 20-30 mL of DPBS to dilute it out
All the best
Melanie
No worries Melanie. Thanks again for your help.
Best wishes
Iker
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