LSK (Lineage-/c-kit+/sca-1-) absolute number determination using FACS - (Dec/23/2010 )

Hi you all!

I'm having trouble determing the total number of Lineage-/c-kit+/sca-1+ cells in mouse bone marrow. I'm currently using BD antibodies. To determine the absolute no of cells I need mostly to find the percentage of lineage- cells but moving slightly the cursor in the analysis protocol the number changes dramatically. Is there any way to overpass this problem so i can be sure that the percentage I take is really the Lin- cells?

nikoletta on Thu Dec 23 11:55:24 2010 said:

You can deplete your lineage with Streptavidin magnetic beads (use Lin-biotin), and compare to non-depleted cells. You will always have many Lin-positive cells left (add Strepavidin-PE-Cy7 to detect them), but it will give you an impression of the Lin population.
Anyway, gating is always relative, so you have to compare to a control. For example, if you have p21 KO mouse, you will compare it to heterozygous and wildtype mouse and set the gate somewhere. Then you should find that percentage of KSL cells using THE SAME GATE differs between mouse lines. That's all you can say.

rsm