Hello, I have two questions :1. if I understand well, you put...

protocol Protocol: Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry
Hello,
I have two questions :
1. if I understand well, you put a 20µl cocktail on a 40µl culot to have a final 60µl staining sample?
2. For me, 3 factors are important for cocktail titration : antibody dilution, number of cells, and the staining volume. Do you check your c
Read more Read less more less
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
1 answer
Sort by: most helpful / most recent

Holden T. Maecker Author Answered Apr 15, 2022

Immunity, Transplantation & Infection, School of Medicine, Stanford University, Stanford

Yes, actually 20 ul antibody cocktail + 50 ul cells = 70 ul total staining volume. We don't check cell numbers, but from 200 ul whole blood, you can expect 500,000-800,000 total leukocytes (including granulocytes). If we do the assay on PBMC, we start with at least 500,000 cells. So 300,000 is a bit low, given losses with washing and acquisition. It would be best to barcode the samples (Fluidigm now sells barcoding kits) and combine them prior to staining, to minimize the loss. We now routinely do barcoding in the context of this assay, as it also saves antibody and increases reproducibility of results across samples that are combined.
0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer