Dear all,
I am isolating PBMC from human blood over Ficoll . I am using 2mM EDTA as a washing buffer. after collecting buffy coat, I washed PBMC 2 times with 2mM EDTA. During washing I set centrifugation parameter 1680 rpm for 15 minutes. but after washing when I count cell. I found cell viability become less than 50%. but I don't understand where exactly I do the mistake. if anyone help to identify the problem, I will be highly benefited.
N.B. I am using LunaTM fluoroscent cell counter to count the PBMC.
Best regard
Razaul Haque
Razaul Haque, washing is generally carried out with PBS. We are using three washing with PBS for PBMC layer, followed by centrifugation at 1200-1300 rpm for 12-15 mins.
Hope it works.
Hello Razaul,
I use PBS to wash cells and then spin at 1400 rpm for 10 min at 4C. Also the faster you work the better yield you get.
Hope this helps
Bhawana Sharma and Kawther K. Ahmed are right. PBS is typically used to wash isolated PBMCs and it is always best to work fast. 7-10 mins of spin is more than enough. If you have been using cold reagents and the steps are being done in the cold, set the centrifuge to 4 degrees. If you are working at RT and your reagents are at RT, set the centrifuge temperature to 22 degrees. Cells don't like sudden temperature shifts. Once the buffy coat was separated we used to do our washes in media (RPMI/FBS or serum free) until the cells were spun down and resuspended in freezing medium. Again, cells are happier in media compared to PBS.
Thank you all for your kind answers. Actually I used 2mM EDTA+PBS to wash media where 2mM EDTA is made in 1x PBS. Still I am not getting the appropriate viability.
Best regard
Razaul Haque
-wash at room temperature with PBS containing 0.5% FBS. If you must use EDTA, omit it from the last wash.
-centrifuge at 300-350xg for only 8-10 minutes. For your particular centrifuge, convert your rpm to xg to see if you are spinning too fast
-your cells might not be dead; try brightfield counting on your LUNA, using trypan blue 1:1 to better see what it is detecting, or better yet have a look using a hemocytomter. Or, try diluting your acridine orange/PI more- apparently different blood cells will stain differently based on concentration or time in AO; F. Traganos and Z. Darzynkiewicz, “Lysosomal proton pump activity: supravital cell staining with acridine orange differentiates leukocyte subpopulations,” Methods Cell Biol., 41 185 –194 (1994). https://doi.org/http://dx.doi.org/10.1016/S0091-679X(08)61717-3 MCBLAG 0091-679X Google Scholar .
Good luck
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