Hi everyone !
I'm trying to get rid CD3+ and CD14+ cells from PBMCs. For that I use CD3 microbeads and CD14 microbeads as well as LS columns (I add both CD3 and CD14 microbeads to the cells and split the samples to load it into several columns to avoid saturation of the column).
When my sample contains 10M of cells, it works perfectly, 80% of the cells are retained in the columns and I have less than 2% of CD3+ and less than 0.5% of CD14+. But, if I have 400M cells, only 60% of the cells are retained in the columns and I have a lot of CD3+/CD14+ cells that are not retained.
I follow exactly the protocol and I did the scale up of the reagents from 10M to 400M accordingly. Did anybody have this issue ?
I tried the LD columns but it killed most of my B cells... one solution that Miltenyi gave me was to put the cells in a second round of LS columns, what are your opinion on that?
Thanks in advance for your help!
hello Rayan El-Kholdi，
In my experience The number of cells that can be combined with the column is limited, about 1×10*8 cells can be combined with a column, so when your target cell number exceeds this number, you need to add new columns, CD3 and CD14 cells in the PBMC itself accounts for a lot, so it is recommended that you increase the number of columns according to the number of cells。The proportion of CD3 positive cells and CD14 positive cells in 400M PBMC is far more than 50%, that is 200M, so you may need to add more columns to improve the ability to capture cells
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