Hi Everyone,
I'm using LSRII for Flow. I have a macrophage population(F4/80+) cells. When I setup the experiment, the PMT voltages of FSC and SSC are perfect with all the cells showing. After compensation controls setup when I run my samples, the cells move off the grid. It seems, the SSC is too high. Therefore the cells shift up the grid and I don't get cells. My cells are fixed with PFA.
Any idea to contain them will be highly appreciated.
Hi Nicole,
Im glad you asked. I should have mentioned. Im doing the unstained with PFA fixed cells but for compensation I use MaxBeads. But there seems no problem with them.
Hi Nicole,
I understand your concern with the compensation. But the beads work fine as they are only meant for compensation controls. The problem lies when the unstained cells used for PMT voltage setup lies well in place whereas the same unstained cells when analysed after compensation moves up the grid with a lot higher SSC. I usually dont analyse the sample in the same day and therefore PFA the cells to fix them for use at a later time. I will try your suggestion to have single cell colour controls. But Im still wondering whats causing the cells to have a higher SSC after the voltages are set. Thanks a lot for your help.
hi amlan,
acquire a full stained sample to set your voltages, then record your beads for setting up compensations. why dont u use beads as unstained sample for compensations?
better to set up the machine and acquire them the same day.
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