hypotonic shock for monocyte+lymphocyte isolation from blood - (Dec/12/2011 )
Hello,
I am a newbie in leukocyte isolation, and I am not sure about one point of the protocol somebody showed to me, just recently.
It was done from mice blood, and in this protocol, after the elimination of most of the red cells by centrifugation in Ficoll gradient with dextran, the few lasting red cells are eliminated by hypotonic lysis 25 seconds in pure H2O followed by return to isoosmolarity and immediate centrifugation.
it seems it is THE classical protocol they use in this lab, but in my previous lab, I used to put hypoosmotic shocks on endothelial cells, they were longer but softer shocks, and they were sufficient to have the endothelial cells swollen and respond by different ways. So I wonder if the monocytes and lymphocytes I want to isolate now are really not suffering from this shock, and we are supposed to use them the same day or the day after the isolation. What do you think about it ? I will work on some mutant blood, if the cells are somehow fragile, we could heve difficulties to isolate them, or we will select a subpopulation, don't you think ?
Kind regards
RR.
Ririiiiiii on Mon Dec 12 15:07:39 2011 said:
Hello,
I am a newbie in leukocyte isolation, and I am not sure about one point of the protocol somebody showed to me, just recently.
It was done from mice blood, and in this protocol, after the elimination of most of the red cells by centrifugation in Ficoll gradient with dextran, the few lasting red cells are eliminated by hypotonic lysis 25 seconds in pure H2O followed by return to isoosmolarity and immediate centrifugation.
it seems it is THE classical protocol they use in this lab, but in my previous lab, I used to put hypoosmotic shocks on endothelial cells, they were longer but softer shocks, and they were sufficient to have the endothelial cells swollen and respond by different ways. So I wonder if the monocytes and lymphocytes I want to isolate now are really not suffering from this shock, and we are supposed to use them the same day or the day after the isolation. What do you think about it ? I will work on some mutant blood, if the cells are somehow fragile, we could heve difficulties to isolate them, or we will select a subpopulation, don't you think ?
Kind regards
RR.
Dear Ririiiiiii,
When I was isolating Neutrophils from human blood using ficoll-paque, we used 0.84% NH4Cl (ammonium chloride) to lyse any unwanted red blood cells (RBC's). We were doing degranulation and chemotaxis assays and found the neutraphils to be readily responsive.
If you use just water then in my humble opinion all the cells will be affected negatively/damagingly by this form of lysis.
Kindest regards
Uncle Rhombus
Thanks for your opinion Uncle Rhombus. I think I will try (NH4Cl) and compare cells' responsiveness. Could you tell me if there is something I have to know about your protocol ? For exemple : 0,84% NH4Cl for how much time ?
RR