Fri Jul 09, 2021 5:30 pm by singh423
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