Basic question on FACS - (Apr/20/2006 )
I am using PE- 2nd antibody for my FACS, but got a weird histogram peak that looks like a half bell shape (the right part). The left is a straight line. The scatter plot looks quite okay. I can not get a normal bell shaped histogram. The parameters are as follows:
FSC E-1, 3.76, LIN
SSC 342, 1, LIN
FL2 875, 1, LOG
I've not done much FACS before. Any comments or suggestions are welcomed. Thanks in advance.
Maybe you gate out the other half of the bell shaped curve?
Try to change/reset your gating settings, maybe worth a try.
Right gates might be a problem, but look at thresholds to, sounds like a treshold issue.
If indeed left of your straight line there is nothing, you probably have set your treshold too high.
Thanks all for your kind reply.
The half bell-shaped histogram remained whether or not I gate the cells.
Yesterday I tried to adjust the threshold. The defalt value in our machine is 52 for all parameters. I tried to adjust FSC and FL2-H individually and simutaneously. It turned out that it's best to adjust both FSC and FL2-H down to zero to get a bell-shaped histogram.
And I can get a perfect histogram for my other cell type without adjusting the threshold, so I think it might be due to the cell preparation. I used 5mM EDTA/PBS to lift up all my cells in order to protect the cell surface receptors. But there were more dead cells in the sample that gave me weird histogram. Another possibility is that these cells are relatively small in size so lower threshold is needed? What do you say?
Acturally it does not matter for my data analysis since I only compare the peak shift. But I am very happy to solve this problem.