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Metal Conjugated Tetramers

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hwtchen

Participant

Posts: 9

Joined: Thu May 07, 2015 6:35 pm

Location: Sickkids, Toronto, Canada

Post Wed Jun 30, 2021 1:23 pm

Re: Metal Conjugated Tetramers

Thanks for the prompt response, Mike! Yes, that is the step I was questioning. I am clearly not spinning long enough to concentrate. I stopped at A2 and left the polymer solution in 4C over night in about 200uL L buffer. I will spin longer to concentrate this morning and target for 95uL L buffer to proceed with A3 and B2 incubation in parallel! Fingers crossed!
Tina Chen
CyTOF Research Project Coordinator, Guidos Lab
CyTOF Operator, Sickkids- UHN Flow Cytometry Facility
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singh423

Participant

Posts: 10

Joined: Mon Feb 12, 2018 10:42 pm

Location: University of Minnesota

Post Fri Jul 09, 2021 5:30 pm

Re: Loss of Signal with Metal Conjugated anti APC/PE antibod

Dear All,
I am trying to optimize my Tetramers staining using Fluidigm anti-FITC_160Gd, anti-APC_163Dy, and anti-PE_165Ho.
The same sample (PBMCs) were labeled with variable doses (0.25, 0.50, 1.0, and 2.0 ul) of Fluorochrome (FITC, PE, and APC) conjugated Tetramers and analyzed by Flowcytomere (Canto II)
The second batch of the same PBMCs was subjected to CyTOF staining (off course with variable doses of Fluorochrome and Metal tagged antibodies (0.25, 0.50, 1.0, and 2.0 ul)
1- Primary staining with Fluorochrome (FITC, PE, and APC), 20minute/4 0C followed by
1- Secondary staining with Metal-tag (anti-FITC_160Gd, anti-APC_163Dy, and anti-PE_165Ho.), 30minute/RT.

The maximum signals were detected at 1ul per reaction with both (Fluorochrome and metal tagged) antibody, however
upon comparing the Tetramer signal I found

1-50% loss of Tet+ CD4 T cells with CyTOF (0.95% PE vs 0.48% anti-PE_165Ho)
2-Over 70% loss of Tet+ CD4 T cells with CyTOF (0.99% APC vs 0.18% anti-APC_163D)
3-No loss in non-specific Tet+ CD4 T cells with CyTOF using anti-FITC_160Gd
Any suggestions and recommendations to improve the signal for both APC and PE.
Thanks and Best Regards
Amar
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singh423

Participant

Posts: 10

Joined: Mon Feb 12, 2018 10:42 pm

Location: University of Minnesota

Post Mon Jul 12, 2021 7:15 pm

Re: Loss of Signal with Metal Conjugated anti APC/PE antibod

Dear All,
I am trying to optimize my Tetramers staining using Fluidigm anti-FITC_160Gd, anti-APC_163Dy, and anti-PE_165Ho.
The same sample (PBMCs) were labeled with variable doses (0.25, 0.50, 1.0, and 2.0 ul) of Fluorochrome (FITC, PE, and APC) conjugated Tetramers and analyzed by Flowcytomere (Canto II)
The second batch of the same PBMCs was subjected to CyTOF staining (off course with variable doses of Fluorochrome and Metal tagged antibodies (0.25, 0.50, 1.0, and 2.0 ul)
1- Primary staining with Fluorochrome (FITC, PE, and APC), 20minute/4 0C followed by
1- Secondary staining with Metal-tag (anti-FITC_160Gd, anti-APC_163Dy, and anti-PE_165Ho.), 30minute/RT.

The maximum signals were detected at 1ul per reaction with both (Fluorochrome and metal tagged) antibody, however
upon comparing the Tetramer signal I found

1-50% loss of Tet+ CD4 T cells with CyTOF (0.95% PE vs 0.48% anti-PE_165Ho)
2-Over 70% loss of Tet+ CD4 T cells with CyTOF (0.99% APC vs 0.18% anti-APC_163D)
3-No loss in non-specific Tet+ CD4 T cells with CyTOF using anti-FITC_160Gd
Any suggestions and recommendations to improve the signal for both APC and PE.
Thanks and Best Regards
Amar
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Aug 26, 2021 3:26 pm

Re: Metal Conjugated Tetramers

Hi Tina,

In some off-line discussions recently, I was looking at amino acid sequences of various avidins.

Wikipedia tells me that Neutravidin is a deglycosylated version of chicken avidin.

Looking up chicken avidin on Uniprot: https://www.uniprot.org/uniprot/P02701
it mentions Cys in the context of an intramolecular disulfide bond, which is between Cys27 and Cys107.

A bit more research and I found this paper in 2003 JBC: https://pubmed.ncbi.nlm.nih.gov/12446662/
They make various chicken avidin mutants, including one called "Avd-nc" with both Cys knocked out. Table 2 in the paper shows a slight decrease in thermal stability compared to wild-type in the absence of biotin (Tm is still 75C, though!), and essentially the same as WT in the presence of biotin.


So, you might give Neutravidin a direction try with Maxpar; it might not work for separate reasons, but it doesn't look like cleaving the disulfide would inherently inactivate it.


Note: Neutravidin has been labeled with metals in some previous Ornatsky papers (eg, PMID 29556738 and 20396648 ), but those experimental sections have few details.


Mike
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