Anti-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] in Flow Cytometry (Human)

NOTE: The data presented in this plot is for the human IgG1 isotype of this antibody.

The plasmids encoding full-length α5 and β1 subunits were transiently transfected into Expi293 α5-/αV- cells (Lin et al., 2022 (Link opens in a new window)) using FectoPro (Polyplus) according to the manufacturer’s instructions. The cells were cultured in Expi293 medium at 37 °C in an 8% CO₂ incubator. After 24 hours of transfection, 3 mM valproic acid and 4 g/L of glucose were added. After 48 hours of transfection, transiently transfected cells were harvested and washed 3x in 1X Hanks' balanced salt solution (HBSS) with 20 mM HEPES pH 7.4, 1% BSA, 1 mM Ca²⁺, and 1 mM Mg²⁺ buffer (labeling buffer) to a final cell density of 1 x 10⁶ cells/mL. In the last resuspension, each sample containing 1x10⁵ cells was resuspended in 50 µL labeling buffer, and the IPI antibody was added to the desired labeling concentration.

Primary antibody: 50 nM in 1X HBSS with 20 mM HEPES pH 7.4, 1% BSA, 1 mM Ca²⁺, and 1 mM Mg²⁺ buffer, 60 min, 4 °C.

Secondary antibody: APC-conjugated goat anti-human secondary antibodies (Jackson ImmunoResearch, catalog 109-135-098, at 1:150 dilution) in 1X HBSS with 20 Mm HEPES pH 7.4, 1% BSA, 1 mM Ca²⁺, and 1 mM Mg²⁺ buffer, 30 min, 4 °C.

Detection: FACS was performed on a BD FACSCanto II. The background mean fluorescence intensity (MFI) was determined using a human IgG1 isotype control (Bioxcell #BE0297) at the same concentration as the primary antibodies. Washing steps were applied after addition of the primary and secondary antibody. Data analysis was performed with FlowJo (Version 10.7.1).