We are using the Ficoll -dextran method for the isolation.We are not getting the white pellet after the addition of RBC lysis buffer. Usually we used to keep for 3 min after the addition of RBC lysis buffer, but by doing like this we are getting only Red pellet. So we added once again and centrifuged it. After that we found that all the cells were dead. RBC lysis buffer pH we set as 7.4, and PBS buffer also 7.4
Can anyone suggest modification for this method
Thank you
I am assuming that your lysis buffer is water? If you leave water on the cells for this long you will kill them due to omosis. My advice is to shorten the lenght of time that the cells are in contact with the buffer. When I isolate these cells I lysis the RBC by inversion in water for 10-20sec and then add in 2X saline to bring the solution back to 1X saline.
We can harvest healthy neutrophils using ficoll paque and dextran method. we also lysis the RBC in water by repeated pipetting for less than 28sec.
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