Can anyone suggest the process of single cell suspension from mouse tumor for FACS analysis? I get it as follows; 1)tumor is minced into small pieces manually 2) transferred to 70-um cell strainer 3) mechanically separated using the plunger of a 5-ml syringe 4)the cells passing through the cell strainer are collected in RPMI1640 media containing 10%FBS 5) Centrifuge and rinse without using Ficoll 6)Stain and fix immediately. The problem of my process is the rate of living cells during FACS analysis is always too low (5~15%). I would like to see surface markers on tumors, monocytes and lymphocytes. It would be most grate if someone could give me any suggestions.
1 is good. Afterwards you could do better. Incubate 1h with collagenase DNAse at 37°C with agitation. Then comes the 70 um filter. 3 washes with PBS+2%FCS+EDTA.Staining without fixation afterwards.
Hi, Kay-Dietrich,
Thank you for the comments.
When I used Accumax including collagenease and DNAse for single cell suspension, cell surface proteins were cleaved though viability rates were slightly improved.
Do you have some know-how to keep surface proteins when using proteinase? I would be most grateful if you could tell me the details.
Thank you so much.
We use Collagenase A (10103586001) and DNase I (11284932001) from Roche in DMEM medium without additives. Stop the incubation after 1 hour with an equal volume (10ml) DMEM+10%FCS. Carefully re-suspend the pellet between washes. Repeat washes until the supernatant becomes clear and is not cloudy (fragments and dead cells). Normally 3 washes is fine. We do multiple surface markers using this protocol. You can find an example on: https://www.researchgate.net/publication/269695276_The_Wilms%27_tumour_suppressor_Wt1_is_a_major_regulator_of_tumour_angiogenesis_and_progression
Article The Wilms’ tumour suppressor Wt1 is a major regulator of tum...
Would this protocol work also with other tissues from mice? I'm specially interested in pancreas, liver, kidney (also bone marrow and peripheral blood but this would be different anyway)...
Tips highly welcomed,
best,
Tom
Sorry, just saw that the tissues are part of your protocol, Kay :-)
However, if you have additional (tissue-related) tips I'd be happy to learn from the experts
All the best and thx for the protocol!
Kidney requires 10min Trypsin extra on the end of digestion to proper digest the glomeruli, for liver it works. Pancreas I did not try. Bone marrow is just cutting the ends of the long bones off and flush the marrow out with a 26g needle; and getting blood is simple.
Hi, Kay-Dietrich,
Thank you very much for the details. Those are very instructive. I'll try next time in accordance with your protocol.
Could you tell me what percentage of living cells do you usually get by the protocol? Do you usually use cold medium when washing? I usually use cold medium during the whole process. I'm not sure why I could not get high rates of living cells by my protocol. Is it because I crashed the tumor manually instead of using enzymes or I used cold medium during the whole process?
I would be mostly grateful if you could tell me the comments.
Regards,
Masahiro Kikuchi
I saw the last question only now. Viability is normally between 60-80%. I wash also cold, important is to stay at the same temperature I.e. work quickly and don't forget to set the centrifuge to 4°C.
Hi, Kay-Dietrich,
Thank you for your comments.
I didn't centrifuge at 4℃. So, I'll do at the same temperature.
Thank you again.