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Anti-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.7] in Flow Cytometry (Human)

Addgene #213660

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 Anti-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.7] (Addgene Antibody 213660) 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 αVβ3 (red). Cells were labeled with the human IgG1 isotype version of the IPI-ITGAV/ITGB3.7 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 3 [IPI-ITGAV/ITGB3.7]
Addgene #213660
APC-conjugated goat anti-human secondary antibodies
Jackson ImmunoResearch #109-135-098

Materials & Methods

Sample

Species
Human
Cell / Tissue Type
K562 cells

Primary Antibody

Name
Anti-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.7]
Source
Addgene
Catalog Number
213660
Species
Rabbit
Gene / Target
ITGAV, ITGB3
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)