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Problem in finding Ly6D , CD127 and FLT+ population

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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Wed Dec 02, 2015 8:53 am

Problem in finding Ly6D , CD127 and FLT+ population

Hello friends,

we are looking for mouse Common Lymphoid Progenitor Compartment (CLP). The general gating strategy to find the CLP is: Lin-FLT3+CD127+ cKit+Sca-1+ intermediate> Ly6D+/-

Regarding antibody staining for Ly6D, CD127 and FLT3 looks fine. when we gate for Ly6D population from CLP we are getting 10% sometimes even less in CyTOF but the same experiment in flowcytometry gives around 60% population for Ly6D and it is acceptable. usually the Ly6D vs cKit population should be 40 :60%.

I think it is because of low staining or may be we need more events, I dont exactly.
any suggestion or help
Attachments
Screen Shot 2015-12-02 at 9.44.49 AM.png
CLP>cKit vs Ly6D
Screen Shot 2015-12-02 at 9.08.24 AM.png
Ly6D vs CD45
Screen Shot 2015-12-02 at 9.22.09 AM.png
FLT3 vs CD45
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Dec 02, 2015 4:28 pm

Re: Problem in finding Ly6D , CD127 and FLT+ population

Hi Komal,

1. Please show us your entire gating strategy, starting from Ungated Found Cells.

2. When you say "the same experiment in flowcytometry gives around 60% population", do you mean you
A) isolated cells
B) split the sample in two
C) ran half on CyTOF, ran half on flow?


Mike
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Thu Dec 03, 2015 7:55 am

Re: Problem in finding Ly6D , CD127 and FLT+ population

Hej mike,

mouse bone marrow cells enriched for cKit.

my colleagues are extensively working on CLP compartment and they analyse the samples in flowcytometry. we use same methods for staining and same clones of antibodies. but I stain the samples for CyTOF. when we compare the results mine differs in percentage of populations and week signal.
Attachments
Screen Shot 2015-12-03 at 8.51.46 AM.png
Screen Shot 2015-12-03 at 8.51.03 AM.png

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