Dear Sagar, macrophages do not proliferate in in vitro cultures. You do not need to fix them for this reason. Anyway, if you want to use any cells capable to proliferate as stimulators (not macrophages), it would be appropriate to irradiate them (25 Gy) or to treat with mitomycin C (Mit C). In these settings formation of synapses with T cells will be possible and you will see primary proliferative responses. In response to fixed stimulator cells you will not be able to see primary responses. You will see responses of antigen-specific memory T cells only. If you want to see proliferation of T cells in response to macrophages, you should take into account that macrophages are weak stimulators of MLR response. Cells used as stimulators should contain dendritic cells. Irradiated (or Mit C treated) splenocytes or adherent fraction of irradiated splenocytes will work as stimulators much better. I hope, enclosed work may help.

https://www.researchgate.net/publication/233960011_Direct_Recognition_of_MHC_Class_I_Molecules_in_Memory_Response_to_Allogeneic_Tumor_Cells?ev=prf_pub

Article Direct Recognition of MHC Class I Molecules in Memory Respon...

Dear Sagar, protocols may quite different in dependence of your goals. I do not understand why do you need to fix macrophages? If this is really required, use 0.1% concentration of paraformaldehyde. In my experiments higher concentrations entirely blocked interactions of T cells with macrophages. Further BE CARE TO REMOVE PFA completely by 4-5 sequential washing.

Objective of my experiment is To study the T cell proliferation in macrophage- T cell coculture.

OK, but you should conceive consequences of every procedure used in your protocol. Fixation will result in inability of macropfhages to establish immunological synapses with T cells. Consequently, you will not achieve optimal activation of T cells. Primary responses will be impossible. It may be, you will see responses after preliminary immunization of animals (donors of T cells) with antigen.

Dear Sagar- I agree with Dmitry that PFA will be a problem. Why not add the monocytes to the flask, add AG for the desired time. The add T-cells to the flask. To determine T-cell proliferation add either Brdu or EDU to the culture overnight. Next day remove T-cells from flask and stain for CD3 (to identify T-cells from any contaminating macrophages. Run on flow cytometer gating on CD3+ vs EDU (FITC) + cells.

Dear Dr. Dmitry and Dr. Daniel

The logic behind the fixation (in my point of view) to stop macrophage proliferation, which may give false results.

Am I write?

Please suggest me the detailed methodology (or attach your paper having the same)

Thanks.

Dear Sagar

For macrophages and lymphocytes co-culture experiments you can use V bottom culture plate. After seeding your cells, you need to centrifuge quickly the plate to keep your cells in contact.

To assess proliferation you can use EDU or Brdu but also CFSE or KI67 stainings. You can find technical information on suppliers website and if you want informations about CFSE you can have a look on Bruce Lyon technical reports available here http://www.cytometry.cn/uploadfile/2007314175023861.pdf

I fully agree with Dr. Dmitry Kazansky.

Dear Sagar, macrophages do not proliferate in in vitro cultures. You do not need to fix them for this reason. Anyway, if you want to use any cells capable to proliferate as stimulators (not macrophages), it would be appropriate to irradiate them (25 Gy) or to treat with mitomycin C (Mit C). In these settings formation of synapses with T cells will be possible and you will see primary proliferative responses. In response to fixed stimulator cells you will not be able to see primary responses. You will see responses of antigen-specific memory T cells only. If you want to see proliferation of T cells in response to macrophages, you should take into account that macrophages are weak stimulators of MLR response. Cells used as stimulators should contain dendritic cells. Irradiated (or Mit C treated) splenocytes or adherent fraction of irradiated splenocytes will work as stimulators much better. I hope, enclosed work may help.

https://www.researchgate.net/publication/233960011_Direct_Recognition_of_MHC_Class_I_Molecules_in_Memory_Response_to_Allogeneic_Tumor_Cells?ev=prf_pub

Article Direct Recognition of MHC Class I Molecules in Memory Respon...

Dear Dr Dmitry,

Thank you so much for your valuable support.

I will definitely consider the points suggested by you.

Thanks

Sagar

You are welcome! Good luck!

Hey Sagar,

I agree with Dmitry. Irradiating macrohage or treating them with mitomycin C is the best approach. However if you use mitomycin C make sure you wash it really well. To induce Tcell proliferation i would use anti- CD3/28 beads rather than the individual antibodies because they require plate coating prior to stimulation.

Splenocytes with anti-CD3 also will be good. Fc receptors on macrophages and dendritic cells bind antibodies via FcRs. This binding to professional APC allows to provide both artificial 1st signal and costimulation for T cell activation.

Hi sagar! May i know which type of macrophages are you using for this assay and what is ratio of Macrophage to Lymphocytes you´r using for the study? Thanks