FAQ  •  Register  •  Login

CD45RA staining issues for smart tube fixed cells

Forum rules
It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
<<

avinash1

Participant

Posts: 16

Joined: Fri Jun 16, 2017 1:59 pm

Post Wed May 05, 2021 3:12 pm

CD45RA staining issues for smart tube fixed cells

Hi fellow CyTOFers

This is Avi from UNC-Chapel Hill. As part of our core services we have been trying to optimize a panel for our clients on SMART tube fixed whole blood cells and get it going on clinical samples. However, we are encountering a CD45RA staining issue.

We have been using a CD45RA HI100 clone from Fluidigm. Thermofisher also mentions that HI100 is resistant to fixation (https://www.thermofisher.com/us/en/home ... tions.html).

1. Has anyone encountered such issues with CD45RA staining on post fixation cells ?
2. If so, which clone works better for post fixation staining of whole blood cells ?

Thanks for your inputs in advance

Regards
Avinash
<<

cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Wed May 05, 2021 3:25 pm

Re: CD45RA staining issues for smart tube fixed cells

Hi Avi

We don't actually use the SMART tubes, but we have found that the H1100 clone for CD45RA works really well after fixing and freezing whole blood with their PROT1 reagent - I think that solution is in their SMART tubes. We tag our own Ab but I think that the Fluidigm reagent should perform similarly. So the problem is not likely the clone but some other aspect of your workflow.


Best of luck
Cynthia
<<

mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed May 05, 2021 3:38 pm

Re: CD45RA staining issues for smart tube fixed cells

Hi Avi,

I'm not clear on what the issue is you're having with CD45RA.

Is it just too dim?
Is it nonspecific?
Fluidigm sells five different CD45RA HI100 conjugates; which one are you using?


I have attached a Layout of 2 donors (WB, ST PROT1-fixed prior to antibody staining). Donor 1 is left, Donor 2 is right, CD4+ on top, CD8+ on the bottom. Note that there's a bit of MSI difference between the 2 donors on both CD4 and CD8; these were stained as one barcoded sample, so the differences should be biology (or processing) rather than staining artifact.


Mike
Attachments
CD45RA signal-PROT1-fixed WB.pdf
(69.91 KiB) Downloaded 188 times
<<

avinash1

Participant

Posts: 16

Joined: Fri Jun 16, 2017 1:59 pm

Post Wed May 05, 2021 4:37 pm

Re: CD45RA staining issues for smart tube fixed cells

Thanks Mike and Cynthia for your inputs.

1. We are using 153Eu tag version of CD45RA (HI100) from Fluidigm.
2. The issue is being "too dim".
3. Majority of the antibodies (both self conjugated and bought from Fluidigm) in the panel titrated so far work but not the CD45RA.
4. We used the same protocol and reagents supplied by SMART tubes to fix the whole blood and lysis for every titration done so far.

I have attached the titration results for your reference.

Regards
Avi
Attachments
Titration_CD45RA.pdf
(181.81 KiB) Downloaded 205 times
<<

eganio

Contributor

Posts: 21

Joined: Wed Nov 07, 2018 8:00 pm

Post Wed May 05, 2021 5:17 pm

Re: CD45RA staining issues for smart tube fixed cells

Hi Avi,

I would bring this issue up with Fluidigm. We've used the HI100 clone successfully on 143 for 8 years. Always gives a robust signal in T-cells. We used to get it from Fluidigm, but now conjugate in-house to the purified MaxPar-ready version of the HI100 clone from Biolegend:

https://www.biolegend.com/en-us/search- ... ibody-9189

Both have worked very well for us in the past. If this is the only antibody that's giving you trouble, there may be a problem with the lot.

-Ed
<<

dianebender

Participant

Posts: 5

Joined: Mon Feb 11, 2019 1:21 am

Post Wed May 05, 2021 5:36 pm

Re: CD45RA staining issues for smart tube fixed cells

Similar to Cynthia's comment...Our users have been using the Fluidigm CD45RA on fixed PBMCs (using SMART tube PROT1 reagent) and have not had problems. I also use this clone on FFPE tissue stained for Hyperion IMC and it works beautifully so I think you have an antibody conjugation issue or workflow problem.

Diane
<<

GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Wed May 05, 2021 10:53 pm

Re: CD45RA staining issues for smart tube fixed cells

Hi Avi,

Similar to Mike, Cynthia, Ed, and Diane, we routinely use CD45RA after Smart Tube fixation and do not experience any problems. We have done at least one careful head to head comparison (same lyophilized antibody in Fluidigm's immune monitoring panel) on the same cells before and after Smart Tube PROT1 and only see a small decrease in staining intensity (~2 fold) with no change in signal to noise.

I would do a head to head test between PROT1 fixed and fresh cells; that should clarify if it's an antibody or a fix issue. Also make sure your are following the SmartTube instructions carefully; I've seen people wash it off (results in under fixation and lysis of many, but not all, cell types), or leave it on at room temperature for too long (leading to over-fixation), as well as incorrect lysis protocols. Any of these could create your problem.

Good luck,

Greg
<<

nsigal

Participant

Posts: 8

Joined: Fri Jul 25, 2014 5:26 pm

Post Fri May 07, 2021 2:41 am

Re: CD45RA staining issues for smart tube fixed cells

Hi Avi,
we use clone HI100 on PFA and smart tube (PROT1) fixed PBMC and whole blood samples, this clone works fine and I have used both in-house conjugated and Fluidigm conjugated CD45RA antibody. Although recently I have to use 1ul of Fluidigm CD45RA on fixed cells when in the past 0.25ul was sufficient. So I think something is happening with Fluidigm CD45RA. I just stained PFA fixed PBMC with Fluidigm CD45RA on 143Nd today and it looked very dim so I have to dig into this problem myself.
But I looked at your titration and it's not just dim, there is no staining at all!

there are couple of things to do, assuming fixation is done properly (and I am sure that my fixation is done properly:-).
1. Go up with the titer, but in your case you already at the maximum 2ul.
2. Compare staining on live vs fix cells.
3. If staining on live cells looks fine but does not look good on fixed cells (and your fixation is done properly,) I would go to Fluidigm and ask them to look into CD45RA titration.
4. If there is no help, do in house conjugation and use it instead of Fluidigm CD45RA. It should work on fixed cells!
I will let you know results of my testing.
Best,
Natalia

Return to Reagents for CyTOF

Who is online

Users browsing this forum: No registered users and 16 guests