I am trying to evaluate neutrophil cytokine production in responses to bacterial PAMPs. I have conducted ICS on monocytes and get great results. Unfortunately, I cannot get a positive stain for IL-8 even with LPS stimulation for 6 hours. I am using a clone that has worked in our lab in the past and can still not get the stain to work. Any thoughts? Protocol: 1. Harvest, wash and block with 10% Ab serum for 10 minutes on ice. 2. Wash and then surface stain with surface marker CD16b 3. Wash and then fix for 35 minutes in PFA/PBS 4. wash and store in PBS O/N @ 4C 5. Perm with 0.5% Saponin 2%FCS in PBS 6. wash and stain with anti-IL-8 (clone E8N1) Ab in perm buffer. 7. Wash and then re suspend in PBS. I am getting a very weak stain in my granulocyte gate that almost appears to be a negilgeable shift (I wouldn't even really call it a shift) in staining when I use whole blood to simulate. Purfied neutrophils still don't respond. As a brief aside, I am purifying using histopaque 1077, collecting the RBC fraction, lysing in ACK and then washing the purified granulocytes. Perhaps this method is making them refractory to stimulation? Thanks!
Maria. I should probably have mentioned that I am adding Brefeldin A. Unfortunately, I still get no signal above my negative control. I am thinking it is either because my neutrophils are activated already or refractory to stimulation? I am seeing this on multiple donors.