Two colour acquisition problem

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Luke F
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Two colour acquisition problem

Hi

I'm trying to set up a template on a FACSCalibur for the identification of T helper and cytotoxic T cells. I'm using whole blood lysis with BD FACSLyse and BDs CD3-FITC and CD4-PE. I have two problems:

1)On the FSC Vs SSC dot plot I can only see the lymphocyte population.

2)On the fluorescence plots I can't see any positively staining populations. I'm using the recommended Ab staining of 20ul per 10million cells.

This may seem like a simple problem but before I continue I wanted a second opinion. Is is just a case of titrating the Ab's i.e adding more, or would the whole blood lysis be at fault? Is there anything I could do to improve this to get other populations on the FSC Vs SSC plot and to give a comfortable separation between the negative and positive staining populations on the flourescence plots. I'm not really a flow expert!!

Thanks for your help!

Omai
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Hi Luke F

Hi Luke F

First, I'm going to move your topic to Immunology where you can get more responses.

Your forward and side scatter have no impact on your labeled populations.
To improve your forward scatter/side scatter, increase the foward scatter to a larger (E) value. Its possible you can only see the lymphocytes because the larger cells are hidden on the right side (too big).

For your labeling, start with a single labeling with increasing antibody concentrations until you get a positive signal. Once both abs are optimized, do the double stain. Make sure you separate your flurophores into FL1 and FL3 so you don't have to worry about compensation (the bleeding of fluorescence from FL1 to FL2 or vice versa).

Hope this helps, come back with any other questions.

Omai

Omai
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bump

bump

Luke F
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Hi Omai

Hi Omai

Thanks for such a quick response! And for moving this to a more appropriate forum.

I did gate on the lymphocyte population before looking at the fluorescence plots so this would obviously affect the number of positive counts. However I couldn't really see a decent positive population on the dot plot or a poitive peak on the histograms.

When looking at the FSC and SSC properties I did change the amp gain and the voltages. I removed a threshold that was present on the FSC, this gave me some natural killer cells along with the lymphocytes, however there weren't any other refined populations, i.e. I couldn't see the monocytes or neutrophils. There were some background events but not enough to define a population.

Compensating shouln't be too much of a problem as it should be fairly straightforward with two colours, however before been able to compensate I should see two separate single positive staining populations, if I instead use anti-CD4 and anti-CD8 (even if they do give slight double-staining results).

I will try it again in the week with increasing Ab concentrations on CD4 and CD8 before I use CD3. If any more advice is given before I start , that would be very much appretiated. I thought that perhaps washing the cells before acquisition might help however there isn't that much background noise anyway. Do you think trying it on two different blood donor samples would help?

Thanks

Luke

Omai
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Hi Luke,

Hi Luke,

I didn't realize you were gating on FSC SSC for your labeled population. I agree this will definitely affect your data.

Two patients sounds like a good idea to me.

I agree that a wash is also a good idea. I always do 3x PBS after my staining.

Omai

(I think some companies offer fixed, labeled blood to test your abs. Maybe BD.)