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

Addgene #209611

Data Submitted By
IPI Staff
Lab Name
Institute for Protein Innovation (IPI)
Submission Date
March 20, 2024
Publication Date
March 25, 2024 (modified March 27, 2024)
Abstract
Experimental results for Anti-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] (Addgene Antibody 209611) in a flow cytometry assay using ExpiCHO-S cells transiently transfected with human RGD-binding integrin α5β1.
Caption: Bar diagram showing mean fluorescence intensity (MFI) from flow cytometry analysis on ExpiCHO-S cells transiently transfected with BFP and either human B7H3 (Control), human RGD-binding integrin α5β1 (human a5/b1), or mouse RGD-binding integrin α5β1 (mouse a5/b1). Cells were labeled with rabbit chimera IPI-ITGA5/ITGB1.2 antibody (#209611) and Alexa Fluor 647-conjugated goat anti-rabbit 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 5 beta 1 [IPI-ITGA5/ITGB1.2]
Addgene #209611
Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific
Jackson ImmunoResearch #111-606-046

Materials & Methods

Sample

Species
Human
Cell / Tissue Type
ExpiCHO-S cells
Additional Information
ExpiCHO-S cells were transiently transfected with BFP and either human B7H3 (Control), human RGD-binding integrin α5β1, or mouse RGD-binding integrin α5β1.

Primary Antibody

Name
Anti-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2]
Source
Addgene
Catalog Number
209611
Species
Rabbit
Gene / Target
ITGA5, ITGB1
Concentration
10 µg/mL
Time
30 min
Temperature
4 ˚C
View in Addgene Catalog

Secondary Antibody

Name
Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific
Source
Jackson ImmunoResearch
Catalog Number
111-606-046
Concentration
1:150
Time
30 min
Temperature
4 ˚C

Additional Information

Additional Information

ExpiCHO-S cells were grown in ExpiCHO expression medium (ThermoFisher Scientific) per manufacturer’s instruction. Integrin α and β subunits were cloned in separate expression plasmids and were transiently transfected into cells together with a plasmid expressing blue fluorescent protein (BFP). Control cells were transfected with plasmid expressing full-length human B7H3 together with the BFP plasmid. Two days after transfection, cells at a density of 3 x 106 cells/mL were labeled with the rabbit chimeric primary antibody.

Washing conditions: The cells were washed twice in 0.5% DPBS, 0.5% BSA, with 0.9 mM Ca2+, 0.5 mM Mg2+.

Detection: Labeled cells were analyzed with an Intellicyt iQue Screener Plus flow cytometer. Cells were first gated for positive BFP expression and then gated for the Alexa 647-conjugated secondary antibody.

Results

Result
Pass (The antibody worked under these conditions)