Pe cy5 mouse anti human cd29 antibody

MDA-MB-231 cells were cultured in 6-well plates until greater than 80% confluence. Control siRNA (4390843, Thermo Fisher Scientific) or ITGB1 siRNA (s7574, Thermo Fisher Scientific) was mixed with MIRFECT (RNAexact) in Opti-MEM medium I reduced serum medium (31985062, Thermo Fisher Scientific) incubated at RT for 30 min and added to cells at a final siRNA concentration of 20 nM. Cell culture medium was changed to fresh cell culture medium without siRNA after 6 h. Cells were washed with PBS twice and dissociated with a non-enzymatic cell dissociation solution for 7–9 min 48 h post transfection. Dissociated cells were incubated on ice with either 100 μL FAM-scr-GreenB1 aptamer (n = 3) or FAM-GreenB1 (n = 3) at 200 nM concentration and 10 μL PE-Cy5 mouse anti-human CD29 antibody (559882, BD Biosciences). After incubation, cells were washed twice with binding buffer and analyzed using an Accuri C6 Plus (BD Biosciences) flow cytometer. Unpaired t test with Welch correction, adjusted for multiple comparisons using the Holm-Šídák method, was carried out using Prism 9.3.1 (GraphPad).

FAM-GreenB1 and FAM-scr-GreenB1 were diluted with binding buffer to 1 μM concentration and folded. MDA-MB-231, MCF-7, and MDA-MB-436 cells were grown in T75 flasks until greater than 80% confluence, washed with PBS twice, and dissociated with non-enzymatic cell dissociation buffer for 7–9 min. FAM-GreenB1 was added at a final concentration of 200 nM along with 20 μL of PE-Cy5 mouse anti-human CD29 antibody (559882, BD Biosciences). FAM-scr-GreenB1 was added at a final concentration of 200 nM along with 20 μL of PE-Cy5 mouse IgG1 isotype control (555750, BD Biosciences). Samples were incubated on ice for 1 h, washed twice with binding buffer, and analyzed using an Amnis FlowSight imaging flow cytometer. Spearman correlation between FAM-GreenB1 and PE-Cy5 mouse anti-human CD29 antibody fluorescence intensities was calculated using Prism 9.3.1 (GraphPad).