Hi!

In principle, you could use the FSC vs. SSC plots to set a gate for your cell population, and double check that it is CD8+. After you measured your sample, among the read-outs you obtain, should be able to know how many total events you acquired, and out of them, how many corresponded to your target cell population (defined by the gates you've set up). Now, the tricky thing is that you need to know in how much volume of sample where those "cells" counted. In some flow cytometers (like the MacsQuant from Miltenyi) you can set up the uptake volume, so for example, if your uptake volume was set up to 100µl, and at the end of acquisition you've counted 10,000 events that fit in your FSC vs. SSC gate and are CD8+ (in order words, your target cells), then you know that your sample had a cell concentration of 100,000 cells/ml.

I am not sure about flow cytometers from other companies have also this feature, or if they indicate you at least how much volume was used for the acquisition; you might need to refer to the user manual.

You can use flow for accurate counting by using trucount tubes provided with lyophilised beads and back pipetting with standard volume from chamber. This is more likely to give you accurate counts. You may stain cells migrated already without affecting the accuracy of the count

Thank you very much for your answers.

Hi Iris Janet Gonzalez-Leal. Probably I used FSC vs SSC plot, to gate lymphocyte. And setting up the number of total gated event for all samples, so I knew the number of Cd8+ cells. I compare these samples based on these number of CD8+ cells. Is it fine? I used BD FACSCalibur, and maybe I could not set up the uptake volume.

Hi Van!

Normally in BD FACSCalibur you set up a number of "events" or "cells" to record, so the cytometer wil be uptaking sample volume until you reach that target. For example, when you have a mixed population, and you want to know how many of them corresponds to a specific cell type, then the preferred approach is to fix your cytometer to uptake a certain amount of cells (from the whole mixed population).

If I understand correctly, you don't work with a mixed population of cells, but rather with pure C8+T cells, right?

Then, in your case, you need a quantitative measurement of how many cells crossed to the other side. So, you need to know to how much volume corresponded the number of cells that you measured. This is why I would prefer a system in which you can "fix" the volume of uptake, rather than taking up sample until you reach a target number of cells. I don´t remember well if the BD FACSCalibur will indicate you how much volume of sample it uptakes for every measurement (try to contact someone from their tech support); if it's not possible to get it, then it would bemore accurate to try Yasser's suggestion.

Hi,

The haemocytometer is not as accurate especially if you have limited number of cells migrating.

For flow cytometry:

Instead of setting limit on events can set it by time on some flow cytometers. I measured T cell migration using FACS. I harvested the cells from the bottom chamber of transwell then washed and stained with T cell antibodies i.e. CD3, CD4/CD8. After staining I resuspended the cells at a fixed volume (i.e. 500ul) then analysed them on FACS Canto. To do this I counted the number of events for 1 minute, then gated on the lymphocyte population (FSC VS SSC) then did histogram of T cell marker (i.e. CD3 FITC) and gated on the positive population and used this as my count. This will give you an indication of the number of T cells migrated. So long as you set up your experiment with media alone vs. media with chemokine can compare the ability of the T cells to migrate.

Hope this is useful.

Thank you very much your reply. I am doing the same method as you suggest. I also count the total number of migrated cells using haemocytometer, then using FACS(stain T cells with CD4/CD8) to count the percentage of CD8+ T cells.To multiple this percentage with total number of migrated cells, I will have the number of CD8+ T cell in the bottom chamber.