Low viability with cryopreserved bone marrow samples
Hello!
I am a just getting started with CyTOF and got stuck already.
I am using cryopreserved human bone marrow samples from patients but I am having poor viability especially after an overnight rest step.
I tried this with PBMCs (no overnight rest though , this is when I was just testing things out) and viability after surface staining was consistently above 80% and usually in the high 80s/low 90s.
I start with ~10 million cells total
I thaw as follows (gentle pipetting all the way):
1) Thaw cryovial quickly (in bath or in my hand)
2) add 1ml of warm benzonase RPMI (20% FBS) slowly and then dump into Falcon with 9 ml warm benz//se media
3) spin down 200G 15mins X1-At this point I have >90% viable cells
4) spin down again 200GX15 min-Viability drops to 70%
5) rest overnight in 20% FBS (no benzonase) (concentration ~3-5 million/ml) in 6 well plates
6) viability next morning is 50%!!!
I could stain with just the surface panel right after I thaw but I 'd like to include a cytokine/intracellular stain so I need to rest them.
On top of that after I use the benzonase free medium I get some clumping and loose a few cells at this step too. So in the end I start with 10 million cells and end up with 2 and lots of dead cells/debris.
I get similar results irrespective of bone marrow infiltration my malignant cells (in case cancel cells are dying easier). Samples have all been stored within the last 2 yrs.
If you have any ideas on how to improve this at any step of the way please let me know.
Many thanks!
Tax
I am a just getting started with CyTOF and got stuck already.
I am using cryopreserved human bone marrow samples from patients but I am having poor viability especially after an overnight rest step.
I tried this with PBMCs (no overnight rest though , this is when I was just testing things out) and viability after surface staining was consistently above 80% and usually in the high 80s/low 90s.
I start with ~10 million cells total
I thaw as follows (gentle pipetting all the way):
1) Thaw cryovial quickly (in bath or in my hand)
2) add 1ml of warm benzonase RPMI (20% FBS) slowly and then dump into Falcon with 9 ml warm benz//se media
3) spin down 200G 15mins X1-At this point I have >90% viable cells
4) spin down again 200GX15 min-Viability drops to 70%
5) rest overnight in 20% FBS (no benzonase) (concentration ~3-5 million/ml) in 6 well plates
6) viability next morning is 50%!!!
I could stain with just the surface panel right after I thaw but I 'd like to include a cytokine/intracellular stain so I need to rest them.
On top of that after I use the benzonase free medium I get some clumping and loose a few cells at this step too. So in the end I start with 10 million cells and end up with 2 and lots of dead cells/debris.
I get similar results irrespective of bone marrow infiltration my malignant cells (in case cancel cells are dying easier). Samples have all been stored within the last 2 yrs.
If you have any ideas on how to improve this at any step of the way please let me know.
Many thanks!
Tax