flow cytometry- mouse spleen cells

6 posts / 0 new
Last post
Beru
Beru's picture
flow cytometry- mouse spleen cells

I am working with mouse T cell. I was using flow cytometry to isolate CD3+ Cd4+ and CD8+ cells from spleen. I had good results until last month when the positive cell number decreased by half. I also used CD90beads-- I got the same decreased cells from the CD90 magnetic sorted cells. I attached my result. The top one are good ones and the bottom one are bad ones.
I appreciate your help.
Beru

Guo-qiang Huang
Guo-qiang Huang's picture
It seems that the percentage

It seems that the percentage of CD3,CD4 and CD90 in the bad files are more resonable than the good file.

marcus muench
marcus muench's picture
There are many things that

There are many things that can be going wrong. I have to say I'm not sure what you were sorting in the top panel, because less than 50% purity of T cells by flow cytometry is not 'Good'.

Some things to consider:
Some cell-surface molecules are prone to capping, CD3 is one of them. You can prevent this by keeping the cells cold and using about 0.01-0.05% sodium azide in your buffer used to stain, wash and sort the cells.

Any problem in the setup of the FACS can lead to poor purity. For instance if your drop delay is incorrect to start with or changes during the sort, then you'll get poor purity.

Make sure the flow process is being watched. A small temporary clog can send all the wrong cells in your tubes (and affect drop delay).

As for magnetic sorting, make sure you are using the correct ratio of beads to cells.

I'm not sure how you are staining or-restaining the magnetically isolated cells, but make sure your not blocking the antibody you use for the final analysis by the presence of unconjugated antibody used to isolate the cells.

It is also worth remembering that your problems may not just be one thing and that a couple of small independent issues may be affecting your results.

Beru
Beru's picture
Thank for your response. I am

Thank for your response. I am optimizing a protocol to sort T cells from mice spleen cells using CD90 microbeads and analyze CD3+ cells using flow cytometry. The total population of T cells in the spleen was 41%(A); and when I do magnetic sorting using CD90 microbeads I got 91% CD3+ T cells (B). Then when I got a new group of mice, the T cell population goes down to 21-25%(C) and magnetic separation gave me only 53% (D) and later 71-75% (E) CD3+ cells. When checking lung swab, actually we isolated Staphylococcus and Bacillus from the old group of mice where the T cells >41% (A). That explains why I got fewer T cell (around 23%) (C) in the new group ("healthy group") but that doesn't help me explain why I got fewer CD3+ cells (also more - 10% B220+ cells) in CD90 microbead isolated cells (D & E). I also have some problem with non stained and stained cells.. (F & G) - linear lined cells in both FL1 and FL2 Vs FL1. I gated those cells and they are all over (H). Thank you for your help.
Beru

marcus muench
marcus muench's picture
I think your problem in the

I think your problem in the new mice is that when you start with fewer of the cells your want, the isolation is less efficient. There are a number of things you may try. First, can you do any additional purification, a negative depletion? Do you perform a light density fractionation? This may help as it reduces cell numbers and removes many of the dead sticky cells that can bind to your beads. You could also try to first deplete cell populations you don't want using beads and then perform the positive selection. This has the benefit that all the cell most likely to non-specifically stick to the beads will be depleted in the first round. You could also try adherence depletion to remove monocyte/macrophages, this is cheap but adds a little extra time to the procedure.

I'm not sure what you are asking about for F & G? I think those data show you have a significant portion of high autofluorescent cells, which are your problem

bminev
bminev's picture
In general the same beads

In general the same beads should roughly lead to a similar purity in the
same type of mouse, but the in the new group it only enriches the T cells by 2-fold. It could be some technicality where the cells were not treated
optimally or the beads got miscalculated and not added enough.

With regard to F and G it looks to me that the cells are not compensated
properly or the antibody has not been added. In general a diagonal line like
that means improper compensation, cells with high auto fluorescence
background (which T cells do not have) or dead cells.