I want to know whether it is a good idea to measure the IFN-g release by intracellular staining (Flow cytometry) from the in vitro culture of naive splenocytes infected with a specific M. tuberculosis antigen for a particular time period say 16-18 hrs? I am planning to do the same under in vivo condition using the tissues from infected mice but I am not sure about the in vitro culture. Can anybody suggest?
Thanks,
Tabasum
You can use Rat ant-mouse mAb conjugated with Alexa or FITC or any other dye.
https://www.pblassaysci.com/content/fitc-conjugated-anti-mouse-interferon-alpha-clone-rmma-1
Juste be sure that your incubation time is sufficient to induce response, I will suggest 24 H in vitro and 3 days in vivo.
The tissue in vivo you use to do need to stimulated before your Flow cytometry assay. We tried the fresh cells from mice infected by BCG, the IFN-gamma is not high. Whereas use the PMA/ Ionomycin along with Bredford A stimulation could enhance the detection. Or you can choose the ELISA kit to measure IFN-gamma in the cell culture supernatant.
When lymphocytes were obtained from lymphatic tissue, apoptotic cells or membrane destroyed cells are frequently observed in comparison to cells derived from peripheral blood.
Destruction of cell membrane make it difficult to quantify the amount of cytoplasmic antigen. My recommendation is to evaluate cell viability after stimulation or to stain apoptotic marker such as 7-AAD prior to permeabilization/intracellular staining step.
Can you stain intracellular protein in combination with surface antigen to classify the lymphocyte into B, CD4(+) T, and NK cells?
Yes you can do. but First you perform a time kinetic study with elisa to see when the cytokine is getting secreted out of the cell. for that add some stimulant in the culture such as mitogen, PMA/Ionomycin or Con A etc. then keep collecting the suparnatant after every 6 hours or so (as convenient) and perform ELISA of all time point to find out the correct time when maximum cytokines are getting secreted.
After that perform your experiment for Flow cytometry. give same stimulation but collect the cells before it secrete the cytokines. for preventing loss of cytokines add Brefeldin A into the culture 1-2 hrs before you collect the cells for FACS processing. Brefeldin A will disrupt the transport mechanism (Golgi) so that you can detect maximum quantity of cytokine through FACS.
All the best
Yeah, you can do it for flow cytometry by following the suggestions given by Noriyoshi and Asad which I would support.
Best,
Kadar Moideen Abbas
In my in-vitro stimulation experiments, I have observed that splenocytes have cytokines release peak between Day 3 and Day 5 after stimulation.
Since you are looking for IFNg response, I assume you are looking at T cell response. For that you need to activate splenocytes with CD3/CD28 antibody as a positive control to compare activation of T cells in splenocyte population with your treatment.
If you are looking for DC responses then PMA/Ionomycin treatment will enhance DC response.
Before analysis you do need to add BrefA to your culture 4 hours prior to intracellular labeling. Make sure you fix the cells first and then permeanbilize cells for intracellular labeling.
I agree with Asad in doing a time point kinetic assay for IFNg release using ELISA or Flow Cytometry before your actual experiment. Just use the proper positive control depending upon which cells you are trying to activate and get a response from.
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