Pacific blue conjugated neo 201 antibody

Analysis of the expression of cell surface and intracellular proteins in purified NK cells and in human carcinoma cell lines was performed by flow cytometry. Cells (1.0 × 10^{6 (link)}) were incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA) in 1 × phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with primary antihuman mAbs in 1 × PBS +1% BSA (Teknova, Hollister, CA) for 30 min at 4°C. Binding of NEO-201 to human carcinoma cell lines was detected by Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA). To detect the NK markers modulated by ALT-803, purified NK cells were labeled with following antibodies: CD56-PE (clone 5.1H11), CD16-PerCP-Cy5.5 (clone 3G8), Tim-3-PE-Cy7 (clone F38–2E2), NKG2D-BV421 (clone 1D11), CD107a-APC-Cy7 (clone H4A3), Granzyme B-FITC (clone GB11), PD-1-APC (clone EH12.2H7), and CD158d-APC (clone mAb 33) (BioLegend). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences). Positivity was determined by using fluorescence-minus-one controls.

Binding of NEO-201 to human carcinoma cell lines was analyzed by flow cytometry. Cells (1.0 × 10⁶) were incubated with 1 µL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1× phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA, USA) in 1× PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4°C. After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined using fluorescence minus one controls. Staining values >10% positive were considered positive for NEO-201 expression. Positive cell lines were ranked according to their quantified expression level (% positive × MFI), and then sorted into groups of low (<200), medium (200–1,000), and high (>1,000) expression.