I used to isolate mouse leukocytes only by using a 40%-80% percoll gradient (550g for 20 min at RT). This technique was very effective to obtain a good and clean leukocyte population to stain by FACS. When I use this protocol I can still find some granulocytes, but I am not sure I can find all of them in my final mixture. For my further experiments I want to be sure I am not loosing them. To do so, I was thinking to perform a discontinuous 40-60-80% percoll gradient with two spins: 500g for 15 min and 700g for 10 min. Is there anyone who has some experience with this kind of isolation? Can anyone suggest me some tips to improve my discontinuous gradient? Many thanks.
Hi Elisa,
Where are you collecting your cells from (spleen, marrow, etc)? How many colors do you have to work with? Unless you're performing a digest of tough tissue (i.e. kidney), I suggest not using a gradient to isolate your cells and instead use either CD45 (if you have a lot of non-hematopoeitic cells) or use a dump channel to get rid of the cells you are not interested in (i.e. CD3e FITC, CD19 FITC to get rid of lymphocytes)
Are you isolating from mouse spleens? We used to do a B-cell prep that was pretty impressive... you might want to reference Virginia Sanders' work...
-Mark
Hi Elisa,
Where are you collecting your cells from (spleen, marrow, etc)? How many colors do you have to work with? Unless you're performing a digest of tough tissue (i.e. kidney), I suggest not using a gradient to isolate your cells and instead use either CD45 (if you have a lot of non-hematopoeitic cells) or use a dump channel to get rid of the cells you are not interested in (i.e. CD3e FITC, CD19 FITC to get rid of lymphocytes)
I am isolating these cells from spleen, lung, liver, uterous and adipose tissue. I digest them to increase the yield of my isolated cells. The problem is that I would like to perform two panels on the same preparation (to compare) , one on my lymphocytes and one on macrophages and eosinophils. My lymphocyte population is so tiny (0.01%) that I need a very clean preparation, and with a 40-80% percoll was perfect. I am expecting to have a lot of macrophages in uterous and adipose tissue together with my little population, but with only CD45 and linage is literally a mess and I cannot see tiny differences. I am planning to do a 9-13 colors flow cytometry for these stainings. Many thanks!
For your messy samples, you can try a DNA stain (i.e. Draq5, Syto Dyes). This will eliminate all the junk fragments (increasing Fsc/ssc) that aren't actually cells but do intrude on all the data plots. You might also want to include a viability dye in case your digestions result in a fair amount of dead cells.
I can definitely try those DNA stains if they can work with my preparation. Thanks! For the viability dye is mandatory as I always need to check my digestion. I have some very good fix able viability dyes from ebioscience to do that.. Thanks a lot!
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