Hello, everyone !
I am trying to isolate CD8+ T cell from mouse spleen. I used CD8a microbeads (Miltenyi, 130-117-044), but the percentage of CD8+ T cell was only 60%. I used Flow cytometry to detect single-cell suspension from mouse spleen and there were too many dead cells. So I think the key problem is to prepare single-cell suspension from mouse spleen which most of cells are live.
Do you have any suggestions about how to prepare single-cell suspension from mouse spleen?
Thank you!
Maybe you could describe what you did and we could give you suggestions?
One method you could try is mincing the (from all surrounding tissue separated) spleen gently between two microscopy glass slides. Do this in a 6-well plate pre-filled with some PBS. Then you perform a digestion with collagenase IV at 37°C (right in the same well) to separate the cells. For time and concentration I would suggest the manufacturer's guidelines, but you can also try shorter times if you are afraid of cell death.
Pipette the suspension 2-3 times up and down with a 1000 µl pipette and transfer through a mesh (for filtering out remaining clumps) right into an eppendorf tube.
All steps should be done very carefully to avoid cell death and don't forget to rinse everything well with PBS in order to not loose too many cells.
Hope this helps!
*Update: PBS + 10 % FCS. And as suggested by the others, red blood cell lysis after having prepared the single cell suspension
We usually perform spleen preps in RPMI 1640 with 10% FCS, having some serum may contribute to keeping the cells healthy. Also, make sure you're doing everything on ice. Performing a shock-lysis of RBCs is also essential, since these vastly outnumber WBCs in the spleen and will run as FSC-lo/SSC-lo with debris and dead cells. Finally, I always advise people to use a viability stain in every phenotyping experiment, because dead cells contaminating your gates can really mess up your analysis.
Good luck!
Up on isolation spleen from mice do the following :
1- rinse the spleen in ethanol 70% for 2 minutes
2- in a sterile 6 well rinse the spleen in pbs ( one well of the 6 well plate) for 5 minutes
3- rinse the spleen in complete DMEM complete medium (10% fbs & 1% penicillin)
4- in a 50 ml falcon tube with orange tip(RNA/DNA free )add corning celll strainer 70 micrometer put the spleen tissue from plate into the strainer
5- with sterile 5 ml syringe filled with 5 ml pbs , pin the spleen with slow infusion wash the spleen while pining the tissue through strainer
6- add 5 ml complete DMEM and centrifuge the cell suspension on 500 g , 4 C for 10 minutes .
7- wash with pbs 3 ml and centrifuge again in 500 g , 4 C for 5 minutes this time
8- resuspend the pellet with 3 ml rbcs lysis buffer with 5 minutes incubation ]
7- centrifuge again on 500 g , 4 C for 5 minutes . and resuspend the pellet in 5 ml complete DMEM .
8- now you have in 15 ml falcon tube spleenic cells suspension
9- then you can use a magnetic beads kit for isolation of your population .
Hope this protocol would help
Hello! I'm having the same issue - too many deads cells in my analysis. I cannot use viability stains because I already have a full panel (6 fluorophores on a FACS Canto). I was processing my tissues using PBS 2% FBS @RT. I see that some of you guys are using cell medium with 10% FBS and on ice. I'll try doing that and see if I have any improvements!
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