cell saparation from lymph nodes and spleen - how can I improve my yield? (Nov/09/2006 )
QUOTE (taliasa @ Aug 29 2006, 01:42 PM)
Hi guys,
I'm isolating APCs and CD4 T cells from spleen and lymph nodes (LNs) by mashing it between 2 frosted microscope slides, isolation of SPMC with Histopaque (or other Ficoll), complement and Abs and CD4 cells by miniMACS magnetic beeds. somehow I'm loosing about 60-65 percent of the cells at the end (after washings by centrifugarion after each step).
what do u suggest I'm doing wrong or can I do to improve my yield?
how can u get rid of connective tissues and any garbage you get after mashing or how can u get a clean suspension after mashing, without any small unmashed remnants (is it the mashing technic?)?
I'm isolating APCs and CD4 T cells from spleen and lymph nodes (LNs) by mashing it between 2 frosted microscope slides, isolation of SPMC with Histopaque (or other Ficoll), complement and Abs and CD4 cells by miniMACS magnetic beeds. somehow I'm loosing about 60-65 percent of the cells at the end (after washings by centrifugarion after each step).
what do u suggest I'm doing wrong or can I do to improve my yield?
how can u get rid of connective tissues and any garbage you get after mashing or how can u get a clean suspension after mashing, without any small unmashed remnants (is it the mashing technic?)?
-taliasa-
QUOTE (taliasa @ Nov 9 2006, 10:41 AM)
QUOTE (taliasa @ Aug 29 2006, 01:42 PM)
Hi guys,
I'm isolating APCs and CD4 T cells from spleen and lymph nodes (LNs) by mashing it between 2 frosted microscope slides, isolation of SPMC with Histopaque (or other Ficoll), complement and Abs and CD4 cells by miniMACS magnetic beeds. somehow I'm loosing about 60-65 percent of the cells at the end (after washings by centrifugarion after each step).
what do u suggest I'm doing wrong or can I do to improve my yield?
how can u get rid of connective tissues and any garbage you get after mashing or how can u get a clean suspension after mashing, without any small unmashed remnants (is it the mashing technic?)?
you could use an homogenizer
It would be easier. you mix the spleen by pushing the piston into the cylinder, and pulling, two or three times, and twisting also. go slowly not to break cells.
you could also use the piston of a syringe and a sieve. you kill hardly cells, but you would have more debris.
after mixing the spleen, centrifuge and put the pellet in fresh medium (5 mL), then let it on ice for 5 minutes, the debris will deposit. keep the supernatant and purify on ficoll.
you can also homogenize the lymph nodes in the same way the lymph nodes.
-Missele-