Anti-mouse IgG F(ab’)2 fragment specific, goat pAb, Alexa Fluor-647 conjugated, Jackson ImmunoResearch, 115-605-006

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  • AF647-F(ab’)2 antibody used in a titration experiment to stain CART19 cells expressing a chimeric antigen receptor or to stain negative PBMCs.
    Ab volumes ranging from 0,5uL to 10uL were used to stain between 5-750.000 cells in remaining volume after washing (2% BSA PBS) (Figure 1). All solution and cells were keept at 4 degrees or on ice and stained in the dark for 30 minutes prior to 2x wash with 2mL 2% BSA PBS.
    A stain index curve were calculated to estimate an optimal antibody volume at around 8uL for 5-750.000 cells (Figure 2).

    High level of unspecific staining seen if cells were washed once, in lower volumes or if washed in plain PBS.

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Western blot
Flow cytometry