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Anti-Integrin alpha V beta 8 [IPI-ITGAV/ITGB8.1] in Flow Cytometry (Human)

Addgene #213653

Data Submitted By: IPI Staff
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: March 20, 2024
Publication Date: March 25, 2024 (modified March 25, 2024)
Abstract: Experimental results for the human IgG1 isotype of the Anti-Integrin alpha V beta 8 [IPI-ITGAV/ITGB8.1] (Addgene Antibody 213653) in a flow cytometry assay with a human sample.

**Caption:** Histogram from FACS analysis on wild-type K562 cells (blue) and K562 stable transfectant cells expressing human RGD-binding integrins αVβ8 (red). Cells were labeled with the human IgG1 isotype version of the IPI-ITGAV/ITGB8.1 antibody and APC-conjugated goat anti-human IgG secondary antibody.
**Image attribution:** Institute for Protein Innovation (IPI)

This report is made available under the Creative Commons Attribution 4.0 International License.

Antibodies Used: Anti-Integrin alpha V beta 8 [IPI-ITGAV/ITGB8.1]
Addgene #213653; APC-conjugated goat anti-human secondary antibodies
Jackson ImmunoResearch #109-135-098

Materials & Methods

Sample

Target Species: Human
Cell / Tissue Type: K562 cells

Primary Antibody

Name: Anti-Integrin alpha V beta 8 [IPI-ITGAV/ITGB8.1]
Source: Addgene
Catalog Number: 213653
Host Species: Rabbit
Target Antigen: ITGAV, ITGB8
Concentration: 50 nM
Time: 60 min
Temperature: 4 ˚C

View in Addgene Catalog

Secondary Antibody

Name: APC-conjugated goat anti-human secondary antibodies
Source: Jackson ImmunoResearch
Catalog Number: 109-135-098
Concentration: 1:150
Time: 60 min
Temperature: 4 ˚C

Additional Information

Additional Information

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).

Results

Result: Pass (The antibody worked under these conditions)