I am collecting whole blood in heparin tubes (green cap) and performing immunofluorescence staining for surface epitopes as well as intracellular cytokines (ICC), being am aware that the 1.8mg EDTA/mL blood interferes with the cell stimulation process. I realised that the commercial RBC lysing buffer we use contains 0.1mM EDTA! I prefer lysing the whole blood before staining as I get better resolution (stain index). Please can someone advise whether the EDTA in the RBC lysing buffer will affect my ICC? Thank you!
I don't think that EDTA in RBC lysis buffer will affect the ICC. Because you may also be washing the lysis buffer with plenty of PBS before proceeding to staining.
Hi,
Both the lysis and the number of washing steps that is carried out before fixing the cells that results in loss of ICC signal.
Initially, we used to lyse the RBC's then stain with a live dead marker followed by staining of surface antibodies. Following that we used to fix and permeabilize and carry out ICC staining.
We found that lysing RBC's with Ammonium Chloride Lysis buffer (which contains EDTA) and then fixing the sample resulted in reduced IFNg and TNFa percentages. We even observed that repeated washing of PBMC's before fixing also resulted in loss of IFNg and TNFa signals. On comparing pre-fixation vs post-fixation we found that the lysis step as well as the washing steps carried out before fixation resulted in loss of the signal.
Now we just add Live dead and Surface antibodies (you do not need to increase your AB conc. Just stain for a longer duration - 45mins to 1 hour) to the whole blood post stimulation and then fix lymphocytes and lyse RBCs in the same step. And continue with the intra-cellular staining.
Alternatively you can lyse RBC's and then suspend your lymphocytes in RPMI supplemented with serum and antibiotics and then stimulate.
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