I am trying to do a lymphocyte proliferation assay but the cells are attaching to the plate. Do I have macrophage contamination?
Hi
What is the method you are using to separate the lymphocytes?
If you are using Ficoll method then there will be monocytes along with lymphocytes.
These monocytes will attach during incubation, but lymphocytes will not. Sometimes you may also find aggregation of cells around adherent cells.
Hey thanks for your response. I am using histopaque 1077. I centrifuge the blood with histopaque to 400 g x 40 min at 18 C. and washed 2 times more with hanks solution collecting the pellet at the end and suspending them in RPMI 1640 media containing non essential amino acids and antibiotics 1X.. After counting them I am plating them in a 96 well tissue culture plate polystyrene and adding Concanavalin A 12.5 ug per well after 24 hours of incubation. I am trying to measure proliferation using alamar blue but I do not see any change in color after 48 hours. I tried to see them in the microscope and I saw a lot of attached cells but as you told me lymphocytes will float. I don't have experience doing tissue culture so I don't know if they are alive and if I should be able to see the floating lymphocytes.
Hi
Currently I do not have experience in Con A stimulated cells, but I have tried using PMA and PHA this also causes the proliferation of lymphocytes. In response to that mitogenic stimuli most of the cells will attach.
Try using U bottom 96 well plate to prevent attachment of cells or you can also use Dyna beads (see Life tech) to stimulate the Lymphocyte proliferation, I got good results by using that beads.
Good luck
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