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HI I really cant find L-Sca-1+Ckit+ cells in CyTOF.

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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 Fri May 08, 2015 6:11 am

HI I really cant find L-Sca-1+Ckit+ cells in CyTOF.

Since so many months I am working with Lineage negative cells to find cKit and Sca-1 cells and lymphoid progenitors, but still I cant find cells.
I have used Miltenys Lineage depletion kit it worked well for Lin-Sca-1 and Ckit+ cells. The miltenys Lineage depletion kit contains CD11b, GR1, TER119 , CD5 , 4-7 and B220.
But I need B220 in lineage depleted cells. So we decided to make our own panel having Biotine CD11b, TER119, CD5 and GR1 from eBiosceince. optimized for best depletion but still cant find cKit and Sca-1 cells in CyTOF. purchased and tried different metal conjugated antibodies for cKit and Sca-1 but still cant find the cells. We are planning to do a real experiment if we find cKit and Sca-1 cells.

Can anyone help me with this problem.

here is the image how it looks for cKit and Sca-1 cells.
Attachments
Screen Shot 2015-05-08 at 8.06.01 AM.png
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri May 08, 2015 5:16 pm

Re: HI I really cant find L-Sca-1+Ckit+ cells in CyTOF.

HI Komal,

If I'm understanding you correctly, you *are* successful using the MIltenyi kit to see your Lin-Sca-1 and Ckit+ cells in CyTOF, but are *not* seeing them in CyTOF if you use your homemade kit. Is this correct? Or have you not looked at cKit and Sca-1 by CyTOF before?

Have you been able to see the B220 cells in regular flow after using your home-made depletion kit? In other words, have you validated that your homemade kit is working properly?


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 Mon May 11, 2015 12:55 pm

Re: HI I really cant find L-Sca-1+Ckit+ cells in CyTOF.

Hij Mikel,

Yes I am successful to see L-cKit+Sca-1+ cells in CyTOF as well as in Flowcytometry if I use miltenys lineage depletion depletion kit

Yes I have done optimization for the home made kit antibodies . for home made kit I could see a nice populations cKit + and Sca-1+ populations and atlease 1% cKit Sca-1 double possitive cells in flowcytometry but not in CyTOF. In CyTOF I am loosing Kit and Sca-1 cells as u can see in the image.


Thanks

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