Abstract
Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.
Materials and Reagents
Equipment
Procedure
Recipes
Technical notes
Acknowledgments
The laboratory protocol was evolved over time in the senior authors’ laboratory using a template that was published in 1986 in Handbook of Experimental Immunology / edited by D. M. Weir; co-editors, L. A. Herzenberg, Caroline Blackwell, Leonore A. Herzenberg. Blackwell Scientific Publications. Institute of Life Sciences is funded by Department of Biotechnology, Govt of India and SP was supported by a fellowship grant from Indian Council of Medical Research.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Thanks Rachel for your interest. Yes, you are right. You could use RPMI for PBMC culture. Our aim was recovery and stimulation of monocytes, so we use DMEM.Thanks
The contents released from a little lysis of RBCs interfere with the monocyte functions.
We usually take PBMC concentration of 1 million cells (a mixture of Monocytes and lymphocytes) and seed 100 ul per well of this suspension in our cultures
Hi,PBMC cells contain monocytes and lymphocytes. We usually grow the PBMC in TexMACS medium which has been designed for high-performance T cell growth, high cell viability, and consistency under serum-free conditions. Cells are then incubated at 37°C and 5% CO2 for 48 hours. The adherent monocytes are removed and the suspended cells are activated with Anti-Biotin MACSiBead Particles and biotinylated antibodies against human CD2, CD3, and CD28. Anti-Biotin MACSiBead Particles loaded with the biotinylated antibodies are used to mimic antigen-presenting cells and activate resting T cells from PBMCs as well as purified T cells in a bead-to-cell ratio 1:2. Expansion is achieved by adding IL-2 and fresh medium every 3– 4 days. Cells are restimulated at day 14 by adding additional loaded Anti-Biotin MACSiBead Particles at a bead-to-cell ratio of 1:2.
Hi The most likely possibility is toxicity of ConA at a concentration of 10ug/ml that you use. Some makes of ConA could be toxic at this concentration. The other possibility is viability of your cells. At the end of 48 hrs you should get >90% viable cells. Hope this helps
I see. I will try proliferation with lower concentrations of Con A. Still the reading would be an accurate reading if I just use WST-1? or is it definitely just accurate with BrdU ELISA or Flow cytometry? Could it be that if I use WST-1 i need to incubate for longer time to have a proliferation reading, since it measures metabolism instead of the DNA synthesis measured through BrdU? As I am very new in the research field, I am truly thankful for your quick reply.
Hi LoannaYou can remove some cells from the plate and can use trypans blue. otherwise you can also stain them by DAPI and check by FACS.thanks
Thank you very much. I have one more question: Do the PBMCs proliferate without mitogen?I seed 150000cells/well and incubate with a plant extract. 20 hours later, i count more cells. Which is the proliferation rate of PBMCs?
HiLoannaMy boss Dr. Ravindran answer this to a previous query. here it is, Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.hope this will be helpful.thanks
Dear Shadia, Can you please clarify why you want to freeze??
I’m purifying the PBMC from human blood and I use the cells right away I’d like to ask if I can freeze the rest of the cells for next experiment, and if I froze them, are they going to be function as the fresh cells if I thaw and use them again and what is the best media for freezing. Thank you for your help and time
We never tried in this way. If I have to advice you based on my experience it is not going to function physiologically equivalent to fresh cells. You can freeze them for DNA isolation or cell lysate preparation but not for any immune response study.
Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.
I activated PBMC with either PHA 5 ug/ml or LPS 1 ug/ml. I used medium IMDM with 20% bovine serum. After 4 days, nothing worked, the cell population decreased! Now I increase the PHA and LPS up to 10 times concentration. I hope it will woek (after the first day, I checked but it still has not been working well). How long is the wait? Any one could provide me suggestions?
Can you clarify when you say 'nothing worked?!' I guess there was no proliferation of cells PHA or LPS. How did you score proliferation? How sure you are about the quality of PHA or LPS? Adding 10 times more PHA or LPS could actually be toxic to cells.
I counted by hemocytometer. Over the time, the cell population decreased. I am using LPS from sigma and PHA ftom Gibco.