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Transcription Factor Buffer Kits

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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Jun 22, 2016 5:29 pm

Transcription Factor Buffer Kits

Hi All,

We have had some issues here with cell yeilds from using transcription factor buffer kit from Tonbo Bioscience. I'm not sure if it is a buffer kit issue or not, but wanted to reach out and ask what people prefered and whether they had similar issues with some kits, but not with others. Once we fix I generally centrifuge at 800xg for 5 min, so I dont feel like we're losing cells because of a lack of spinning. In some cases, though, we notice we are only getting 10-25% of our cells back. Any thoughts?

-Chad
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afs1990

Participant

Posts: 12

Joined: Wed Jul 29, 2015 4:09 pm

Post Mon Jun 27, 2016 3:11 pm

Re: Transcription Factor Buffer Kits

We were using the Tonbo transcription factor in the past, but I can't say we ruled out if that was causing any weird problems. We found that we were losing >95% of our cells after fixation, but it turned out to be the staining buffer prefix that was causing problems.

It was a very weird issue. We switched to using a FBS based staining buffer to a BSA based staining buffer, both of those buffers made in house. The Tonbo kit worked without any problems using the FBS based staining buffer. On our first run using the BSA staining buffer, I ran an aliquot of those cells pre-fixation on a regular Fortessa to make sure I had cells and that my fluorescent antibodies stained properly before staining with secondary antibodies. I did not notice any irregularities when I ran those cells when using that BSA staining buffer pre-fixation. However, after washing out the iridium, I lost most of my cells. It took us a couple of experiments to pinpoint that our BSA staining buffer was causing the problems.

What staining buffer are you using to stain pre-fixation?

Have you been noticing any clumping issues?

In the end, we ended up switching to using the BD BSA staining buffer before fixation and ebio foxp3 fix/perm after fixation, because that was the buffer set our collaborators were using.

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