Cell population using spectral flow

[Ka Ka Yu]

Dear TooT, 

I am currently trying to characterize my cell populations in Day 25 BMO derived from KOLF2-C1 cell line passage15. I have used the Nat protocol paper method. 

They were grown in Aggrewell plates on Day -2. Note, revitacell was not added as KOLF2 seem to grow fine without. On Day0-day3, in 5% O2 and 3uM CHIR 99021, the following days were same as the protocol with the same cytokine concentrations. I could not detect CD45+ populations. The BMOs did have erythroid cells. They were also biased towards stromal populations, endothelial cells and MSCs. 

In your experience, do you think it is to do with faulty cytokines?

Thanks so much for your help! 

Ka Ka

[Abdullah Khan]

Hi Ka Ka, 

KOLF2-C1s should give a nice big CD45 cloud - looking at your flow, I think you have a viability problem. How are you digesting this? Can you see releasate in the cultures (shoudl be a nice cloud of round haem cells). 

Abs 

[Ka Ka Yu]

Thanks for your suggestion. Attached is a photo of the organoid under microscope at Day 10. Are the orange arrows pointing to the haem cells? See attached. 

I forgot to mention that I also did spectral flow cytometry at Day 13 and Day 20 from the same batch mentioned above, viability at 80% and 60% respectively. Both did not show any CD45+ fraction. Attached is my data for that. 

I am digesting them using 20mg/mL Collagenase Type II (merck, C2-22-1G) diluted in HBSS. I will add this solution to the organoids and incubate in water bath 37C at 5min intervals until pellet no longer visible and completed dissociated. Maximum time in collagenase was kept below 30mins. The organoids digestion was stopped using stopping buffer (PBS, 1% FBS, 20mM EDTA). 

Thanks for your input!

Ka Ka

[Abdullah Khan]

Hi Kaka - we've switched to using Col D (comBO paper) for the digestions - this is much better for viability! 

Abs