i found a lot of auto-fluorescence for flow cytometry on the dendritic cells.
The person in the flow cytometry facility is a newbie who cannot help me set up a compensation protocol. He complained that my cells have auto-fluorescence which screwed up everything. I wonder whether anyone might have experience in handling such cells and could help me on how to set up a good compensation protocol.

I prepared the Dendritic Cells with prepared CS-GSF and IL-4 stimulation of monocyte. i am using 4 color staining.

Thanks.

I assume the difficulty is that you have wide range of autofluorescence. Your isotype control probabaly is a long diagonal (on dot plots) over a decade of your plot.

There are several things you could do. You could try using bead to set your comps after adjusting the proper voltages using your cells.

If you use your cells, then there are tow things you need to pay attention to. One is that the marker you use to comp may be only expressed on a subset of cells. If it is expressed on the mature cells, then you want to set it to even out with the highest autofluorescencent cells and visa-versa. You may want to look at the forward and side scatter provile to see if you have a spread of low and high side scatter cells. I would gate on the low side scatter cells to have the cleanest population to work with for setting up the machine.

Second, make sure you block non-specific binding with mouse serum or an appropriate source of unlabeled antibody. Even so, when you set up your comp tubes, add isotype control for the three colors that you are comping against. HLA-DR is a good bright marker for comp, so HLA-DR-FITC, isotype-PE, isotype-APC etc would be one tube. If you leave out the isotype, then the lack of nonspecific binding may cause the background to fall significantly below your negative control (isotype control, not unstainined). Then you don't know how far to comp. This can be a bigger problem when working with cells like DC and monocytes compared to lymphocytes. Also, you may want to invest in a good birght marker, like DR, in all four colors (3 colors + PI?) to help with the compensation.

Another option is to use some other cells to help you comp. Set the voltages using your DCs, then use fresh PBL or even a cell line using the SAME voltages. As long as the cells are visible on your dot plots, you can mark off were there negative control lies and then set your comp with these cells. In a sense, I'm just using some other cells as beads.

Hope that helps.

how do you adjust voltages ?
we adjusted the voltages to very low so that the unstained cells are within 0-1. But when we then ran the beads using these voltages , the beads show very weak signals because the voltage is too low.