AlexaFluor 647 conjugated Mouse anti HLA-2 antibody, clone BB7.2 used to assess HLA-2 expression on SupT1 cells by flow cytometry,
Image caption:
Antigen-specific elimination of target cells by sTCR-Saporin as measured by flow cytometry.
(a) SupT1 cells expressing the indicated SCT were mixed at a 1:1 ratio with HLA-A2neg SupT1 cells and co-cultured for 3 days in the presence of relevant or irrelevant sTCR conjugated to Saporin, as indicated. At end of culture, cells were stained with anti-HLA-A2 AF647 to determine the frequencies of HLA-A2 positive and negative target cells among FSChi,SSChi events using flow cytometry. (b) Assay was performed as described in (a) and the percentage of SupT1 cells expressing indicated SCT that was eliminated by addition of sTCR is shown, calculated as described in materials and methods. Error bars indicate SEM of n = 3. (c) HLA-A2pos SupT1 cells mixed at a ratio of 1:1 with HLA-A2neg SupT1 cells were incubated overnight with indicated concentrations of peptide, washed, and indicated sTCR-Sap was added. SupT1 cells expressing SCT were used as positive controls (disconnected symbols). Culture and analysis to determine the percentage of eliminated HLA-A2pos targets was performed as described in (a,b). Results shown are representative of two experiments performed, and error bars represent SD of duplicates.
From: Walseng E, Wälchli S, Fallang L-E, Yang W, Vefferstad A, Areffard A, et al. (2015)
Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery.
PLoS ONE 10(4): e0119559.
AlexaFluor A647 conjugated Mouse anti HLA-2 antibody, clone BB7.2 used to assess HLA-2 expression on SupT1 cells by flow cytometry,
Image caption:
SupT1 cells, which are HLA-A2 negative, were transduced withSCT-M1, SCT-CD20 or SCT-EBV constructs, respectively. Cells staining positively with anti-HLA-A2 were sorted, expanded and stained with anti-HLA-A2 antibodies conjugated to Alexa Fluor 647 (anti-HLA-A2-AF647), as shown. Untransduced SupT1 cells (filled grey) were used as a negative control.
From: Walseng E, Wälchli S, Fallang L-E, Yang W, Vefferstad A, Areffard A, et al. (2015)
Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery.
PLoS ONE 10(4): e0119559.
Figure A. Alexa Fluor 488 conjugated Mouse anti Human CD3 and Amethyst Orange conjugated Mouse IgG2b isotype control . Figure B. Alexa Fluor 488 conjugated Mouse anti Human CD3 and Amethyst Orange conjugated Mouse anti Human HLA-A2 . All experiments performed on human blood gated on live single lymphocytes, in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer
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