I’m trying to isolate only peripheral lymphocytes from whole blood using histopaque media. for that I put 3 ml blood in a 15ml tube and layer 3ml histopaque on it. Then i centrifuge it at 200g for 30 minutes. I get a white layer, apparently the buffy coat. But it also contains other cells, how do I only isolate lymphocytes from whole blood? I have also attached pictures of isolated buffy coat, kindly confirm if it’s fine?
Dear Mehar
firstly when separating mononuclear cells from whole blood using density gradient method (e.g. ficoll) you usually need to dilute part of the original sample with other media (e.g. 1:1 dilution with PBS). In your case you must see the specifications in order to see if dilution is required.
After that you need to add your density gradient medium in a new centrifuge tube (according to the specifications of the medium) and then carefully add the diluted sample (if dilution is needed in your case) on top without mixing (very important)!
After centrifugation you should be able to obtain the buffy coat (intermediate-layer/interphase between density medium and plasma) that contains both lymphocytes and monocytes.
The exact protocol should be provided along with the density medium by the provider.
For further lymphocyte separation you might need other density gradient media of specialized kits...!!!
Okay thanks Sir.And kindly tell after isolating the PBMCs, we need to suspend it in RPMI-1640 media after washing with PBS, plz confirm whether RPMI-1640 is enough for the growth of lymphocytes or do I need to add any other supplements like FBS in it? If so, it what ratio. Kindly help me out here, I couldn’t find literature on the internet.
The RPMI-1640 is more enriched than other media.
If you have the product that is describes at the following link:
https://www.thermofisher.com/order/catalog/product/11875085
then you probably need to add 10% FBS (Fetal Bovine Serum), some antimicrobial agents and potentially a mitogenic factor like phytohemagglutinin (PHA). But you have to search that on your own more carefully because there is enough bibliography.
I also provide you with a research paper that describes the formula for the medium and the handling of Human T Lymphocytes:
Article Human T Lymphocyte Isolation, Culture and Analysis of Migrat...
I hope that was helpful
Best regards
Antonis Tsintarakis Sir, thanks a lot. I have attached the picture of the RPMI-1640 media I will be using For culturing lymphocytes.
can I alternatively use this media gibco karyomax media in colcemid solution(prepared in HBSS)?
No you shouldn't use that medium cause it contains colcemid and will cause cell cycle arrest to your cells!!! It is used for other purposes such as karyotyping and it retains cells at the metaphase stage. You don't want that.
Use the previous medium you mentioned! It is used specifically for PBMC cultures...
Antonis Tsintarakis okay but will the RPMI-1640 that I have attached above be sufficient alone for growth of lymphocytes?
With some FBS added I would expect so, the expiration date put on reagents are super conservative, and in many case ridiculous, as I've even seen such dates on a bottle of sodium chloride!!!
Ok, so if i want to standardise this isolation technique for LIT(Lymphocyte immunization therapy)therapy, how much blood volume would i need and if I can use normal saline for blood dilution?