High autofluorescence in cultured cells - (Jun/05/2008 )

When I analyse cultured bone marrow cells using flow cytometry I have what I think is a lot of autofluorescence, which makes it difficult to analyse the data unless you either set the quadrants beyond the second log on the dot plot or reduce the FL1 and FL2 voltage significantly.

Is there a way to get the plot with unstained cells to look more normal (ie grouped more or less within the first logs) instead of being a long, diagonal streak? The attached file is a sample of unstained cells where I have reduced the FL1 and FL2 voltage. If I didn't, there would be a diagonal streak across most of the plot.

The cells are immature bone marrow cells that were plated in methylcellulose media and have been cultured between 8 - 14 days . I isolate all the cells on the 35 mm petri dish by rinsing it with PBS/1% FBS and washing the cells 2-3 times before starting the staining protocol.

The gate I am using in that plot is the same as in the other, subsequent plots and I used 7AAD for dead cell exclusion, although not in this plot. The high FL1/FL2 autofluorescence is still there in the form of a diagonal streak.

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when you say - significantly reducing FL1/2 voltage......how much by? we culture HEK cells with and without transfectants, and where they are 'autofluorescent' to start with, after culture, we have to turn the voltage down further (this is using DiVa.....) but never lower than 400 (well 380 at a push) for FITC. FSC/SSC values are somewhere in the region.......50/150.......

your FSC/SSC looks fine, but then is does depend on how far you are bringing the voltage down by.

Decreasing voltages is fine as long as you are in the linear range of your photonmultiplier, settings can vary quite a lot between different cells, or even intensities of expression of you're labelling. A colleague of mine has a certain expression vector for EGFP and we have to turn down PE and FITC-channel voltages because otherwise the EGFP signal is impossible to compensate for (she uses the same cells for other exps with "normal" voltages). So I wouldn't be worried about lowering voltages too much.
Some questions that might help:
1. Can you give different colours to your quadrant and see where your "autofluorescent" cells are to be found on your FSC-SSC plot? Maybe there you could gate them away?
2.Or do you have other channels left where you could gate away autofluorescent cells?
3. do you have a fluorescence microscope? If for some reason there's a problem with your cell culture, some cells might be autofluorescent and you would notice this.

I can gate away some of the autofluorescence, but not all of it. The problem I have with the fact that I'm reducing the FL1/FL2 voltage is that the stained populations shift as well so that they are sometimes almost off the plot.

The cultures look healthy under a regular microscope, but I haven't tried looking under a fluorescent microscope.

After gating away your autofluorescence, what percentage of "double" positive cells do you get? If very low, you would see them on a dot plot, but they will not interfere with your "real readings". Can you post a density or contour plot of "gated away" vs "not gated away" (if possible with percentages for each quadrant.
What other FL's do you have?

When lowering your voltages, obviously all your cells (both stained, autofluorescent and unstained) will move towards yourX X-Y-axis intersection. This is not a problem as you can also move your quadrants in that direction, so as long as you take with you negative (isotype) controls and single stained controls (for compensation settings) you should be able to distinguish between unstained and stained cells.