mouse splenocyte preparation - (Oct/16/2000 )
Spleenocytes are very sensitive to manipulation. In my hands I even don't lyse erythocytes as the viability of the cells drops sharpely. I usually isolate then by cutting the spleens in small fragments with a blade and then smashing with a seringe plug. Then I let setle the debris in a 15 mL falcon tube and recover cellular suspention. Viability is elevated.
I'm working on mouse spleen DCs. I have a BIG problem with splenocytes : I have always a lot, a lot of died cells in my suspensions. after an overnight culture, about 50% are dead. Have you got any solutions for this ? did you already have this problem ?
thanks a lot
I've only got 40 micron cell strainers in the lab. can they be used for splenocytes too?
i used to collect splenocytes by flushing medium into spleen with the help of needle and syringe. at the end u will have splenocytes in the medium and bulk of connective tissue which will turn into whilte color since it is depleted of splenocytes and RBC remains intact. collect the medium and leave for minutes in falcon tube, so that incase there is any debris it will settle down or you can go for a low speed (1200rpm) spin to settle down the debris.
further you can proceed for RBC lysis or culturing.
hope it will help you
you are welcome for further clarifications
you may not use mesh,spleencell is easy to separate ,debris can sediment itsetf only put minutes,you soak up
rolleyes: splenocytes isolation do not need mesh to remove derbris. Accronding my experience, two treatments of homogenize and RBC lysis buffer treatment cause debris during splenocytes isolation.
first, you just stat the collector under room temperature for 1-3 mins and to collect supernatant, which can used to remove connective tissues and debris.
Second, you can prepare the column to remove debris, you just prepare a little bit cotton and put it to the neck of pasterpipet, then treated with autoclave, this column are ready for use. You just put the solution containning splenocytes into the column and debris would be trapped in cotton.
Of course, if you are working for a rich boss, you can buy 70 or 100 micrometer (FAlcon)of mesh to remove the debris during splenocytes isolation
I'm isolating APCs from spleen by mashing it between 2 frosted microscope slides and isolation of SPMC with Histopaque (or other Ficoll) and Abs. in the last three experiments I'm loosing almost all cells after washing the sup applied from the Histopaque separation.
what do u suggest I'm doing wrong or can I do to improve it?
by the way, how much Histopaque do I add to the suspension I recieved after mashing the spleen and for how long should I centrifuge it?
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?)?