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phosphlow p-STAT5

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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Wed Dec 11, 2013 2:11 am

phosphlow p-STAT5

Hi All,

I seem to be getting high background as well as inconsistent results with p-STAT5 staining. The stim is 10 minutes with a high dose of IL2. This has been very consistent for us in a standard flow assay. We usually use thawed human PBMC and can get a 10 - 50% positivity in CD4 cells. With Mass Cyto., I'm only seeing a small shift with a 2-10 percent frequency. However, on some days it's brighter and higher. Is it necessary to fix after the intracellular stain and before the Ir labeling? Could it be from adding too many surface antibodies with the stim? Are there some tricks with mass phosphlow?

Regards,
Ian
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Dec 11, 2013 4:20 pm

Re: phosphlow p-STAT5

Hi Ian,

When you say "adding too many surface antibodies with the stim", what do you mean? Are you adding surface antibodies during your stim, and then fixing everything at once?

The general workflow for phosphoCyTOF for many people at Stanford is:

1. Thaw (if PBMC)
2. Rest (if PBMC)
3. Stim (15min is most common)
4. Fix (PFA is most common)
5. Surface
6. Perm (Methanol is usually required if you're going after STATs)
7. Intracellular
8. Fix (second fixation is usually advised; usually PFA)
9. Ir intercalator
10. Wash.

Steps 8 and 9 can often be combined so second fixation and Ir are done in a single step (1.6% PFA and 0.2x intercalator into PBS, 4C, often overnight).


Mike
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ErinSimonds

Master

Posts: 50

Joined: Tue May 13, 2014 8:04 pm

Post Tue May 20, 2014 7:16 pm

Re: phosphlow p-STAT5

Hi Ian,

pSTAT5 is usually very robust by both flow and mass cytometry. I'd suggest splitting a sample and comparing side-by-side on flow vs. CyTOF, putting the cells through all the same steps. This will tell you for sure if the sample preparation is the issue. Detailed protocols from the Nolan lab are available at:

http://cytobank.org/nolanlab/experiment_protocols/

In the "Mass Cytometry Protocols" section, there are several modular protocols. For the phospho-STATs, you want to use modules 1a, 2, 5, and 6. As Mike said, post-fixing is done routinely (it's module #6). It isn't absolutely necessary, but it helps.

If the side-by-side comparison above suggests that sample preparation is not the issue, then it is probably the antibody. If you bought it from DVS, contact them and let them know. If you conjugated it yourself, it may not be working.
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Thu May 29, 2014 3:12 pm

Re: phosphlow p-STAT5

Hi Ian, we routinely get good STAT5 staining with a custom ab from Cell Signaling (Tyr694) (C71E5) conjugated to 156Gd.

To test the staining, we stimulate warmed human whole blood with IFN-alpha for 15 minutes at 37 degrees. The staining has been good and reproducible and is reflected in our side-by-side flow analysis.

We do methanol perm and make sure we really wash out the methanol before adding the intracellular antibodies.

We incubate the antibodies at room temp for 1.5 hours or overnight at 4 degrees. I also do 3 PBS washes and 1 water wash before running these samples.
I found that resuspending the cells in a fix/perm buffer for subsequent Ir staining can be useful if Ir staining is weak, was that a problem for you? I know others do the Ir and intracellular staining at the same time with success, I can't remember if that was with phospho staining though.
Rob

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