Good evening,
I want to separate lymphocytes from human blood then culture these cells for stimulation and determine the fractions of T, B and NK cells in samples.
Is the attached protocol is the suitable one?
also about PHA, Is PHA-M a good choice in this protocol?
finally, Can I skip addition of IL2, or it can be substituted with IL6 or IL17? If it yes, what is the conc needed?
Thanks in Advance.
Hello Esraa Mohsen
1. Is the attached protocol the suitable one?
Yes, you may use this protocol, but it seems to be incomplete. What about the fractions of T, B and NK cells in the samples?
You may use immunomagnetic cell sorting (MACS) to separate various leukocyte populations. Please refer to the article attached below.
https://medicine.uiowa.edu/pathology/sites/medicine.uiowa.edu.pathology/files/wysiwyg_uploads/LRS_ART_5_Jl_Immun2005.pdf
2. About PHA, Is PHA-M a good choice in this protocol?
Earlier nomenclature of phytohemagglutinins was based as per the purification level, for instance, PHA-M, was named after residual content of mucoprotein, whereas PHA-P indicated pure protein. However, the recent nomenclature refers to biological characteristics of phytohemagglutinins, for instance, PHA-P preferably agglutinizes erythrocytes, and is now termed as PHA-E. PHA-M was similarly re-named to PHA-L, due to its particularly stimulating effect on lymphocytes. So, I would recommend the use of PHA-L in the protocol as it is the appropriate PHA with lymphocytes stimulating properties.
3. Can I skip addition of IL-2, or it can be substituted with IL-6 or IL-17?
No, you cannot skip the addition of IL-2 or IL-15. You may use IL-6 but along with TNF-alpha.
IL-2 was first described as a growth and survival factor capable of inducing T cell proliferation, and the elimination of IL-2 from proliferating T cells can lead to cytokine withdrawal-mediated cell death. IL-2 is necessary for the development of T-cell immunologic memory as well as induction of regulatory T-cell activity. In addition, IL-2 facilitates the production of immunoglobulins by B cells and induces the differentiation and proliferation of natural killer (NK) cells.
IL-2 and IL-15 share many functions in regulating both adaptive (stimulation of T cell proliferation and induction of cytotoxic T lymphocytes) and innate (activating natural killer (NK) cells) immune responses.
Therefore, T cells may be activated in an antigen-independent manner by using cytokines IL-15 or IL-2 alone, or you may use a combination of IL-6 and TNF-alpha.
Please refer to the link below.
Article Cytokine stimulation of T lymphocytes regulates their capaci...
Best.
Thanks alot Dr. Malcolm Nobre for this answer
I am really appreciate it.
About IL2 or IL6 plus TNF-alpha
Is these reagents used for T-cells specially activation and proliferation?
I need to proliferate B-cells and T-cells also to detect the effect of specific drug on them. what can I use for this?
Lymphocyte separation and culturing technique is a method used to isolate lymphocytes from whole blood. The typical process for carrying out lymphocyte separation is the density gradient centrifugation. Centrifugation is the commonly used method for processing blood so that the cells and particles of the same size, shape, and density sediment as separate zones without convection.
Once you have isolated the lymphocytes, you can culture them using a variety of techniques. One common method is to use RPMI 1640 medium supplemented with fetal bovine serum (FBS) and antibiotics. The cells are then incubated at 37°C in a humidified atmosphere containing 5% CO2.
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