Anti-Integrin alpha V beta 6 [IPI-ITGAV/ITGB6.4] in Flow Cytometry (Human)

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

K562 stable transfectant cells expressing the appropriate full-length RGD-binding integrins were cultured in RPMI 1640 with 10% FBS and 10 mM HEPES at 37 °C in a 5% CO₂ incubator. On the day of the experiment, K562 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 cell/mL. In the last resuspension, each sample containing 1 x 10⁵ cells was resuspended in 50 µL labeling buffer, and the IPI antibody was added to the desired labeling concentration.

Washing conditions: Cells were washed 3x in 180 µL of 1X HBSS with 20 mM HEPES pH 7.4, 1% BSA, 1 mM Ca²⁺, and 1 mM Mg²⁺ buffer at 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).