Goat anti human apc fab igg

GBM Tumorspheres were dissociated using 0.2 Wünsch unit/mL Liberase Blendzyme 3 (Millipore Sigma, Cat#5401119001) plus 10 μL DNase (Worthington Biochemical, Cat#LK003170) and adherent cultures were dissociated using dissociation enzyme TrypLE (ThermoFisher, Cat#12605028). The single cells were resuspended in PBS+2 mM EDTA (Invitrogen, Cat# AM9260G). Cells were then stained with APC conjugated mouse monoclonal human Carbonic Anhydrase 9 antibody (1:10) (R&D, Cat#FAB2188A) or a matched isotype control and CA9 DATEs followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170) and incubated for 15 minutes at room temperature. T cells were stained with CA9 DATEs (15 minutes RT) followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170), anti-CD25 (Miltenyi Biotech, Cat#130-113-283) and anti-CD69 (BD Bioscienecs, Cat#555533). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, Cat#A07704). Compensation was performed using mouse IgG CompBeads (BD Biosciences, Cat#552843). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter) to assess the level of CA9 surface expression.

The specificity of CA9 DATE for GBM cells, ccRCC cells and T cells were tested using flow cytometry analysis. CA9^hi GBMs, CA9^- GBMs, ccRCCs and T cells (isolated from human PBMCs for GBM study and Jurkat cells for ccRCC study) were resuspended in PBS plus 2 mM EDTA and were stained with CA9 DATEs followed by the secondary antibody, goat anti -human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170) staining. GBM and ccRCC cells were incubated for 15 minutes at room temperature and for 20 minutes on ice, respectively followed by 15 minutes incubation at room temperature for the secondary antibody staining. Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, Cat#A07704) and samples were run on MoFlo XDP Cell Sorter (Beckman Coulter) to assess the level of CA9 DATE binding to each of the above-mentioned lines.