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Flow cytometry for Anti-Integrin alpha 4 [IPI-9C10]

Addgene #213670

Data Submitted By
Zachary Anderson , Haisun Zhu , Travis Riedel , Deborah Moshinsky and Rob Meijers
Lab Name
Institute for Protein Innovation (IPI)
Submission Date
May 24, 2024
Publication Date
June 12, 2024 (modified June 12, 2024)
Abstract
Experimental results for Anti-Integrin alpha 4 [IPI-9C10] (Addgene #213670) in a flow cytometry assay with a mouse sample.
Caption: Histogram from FACS analysis on ExpiCHO cells transfected with mouse integrin α4β7 (red) or B7H3 (gray). Cells were labeled with Anti-Integrin alpha 4 [IPI-9C10] (Addgene #213670) and Alexa Fluor 647 conjugated affinipure F(ab')2 Fragment Goat Anti-Rabbit IgG secondary antibody (Jackson ImmunoResearch, 111-606-046). Labeled cells were analyzed with an Intellicyt iQue Screener Plus flow cytometer.
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 4 [IPI-9C10]
Addgene #213670
Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific
Jackson ImmunoResearch #111-606-046

Materials & Methods

Sample

Species
Mouse
Cell / Tissue Type
ExpiCHO
Additional Information
ExpiCHO cells were transiently transfected with B7H3 (negative control) or mouse integrin α4β7.

Primary Antibody

Name
Anti-Integrin alpha 4 [IPI-9C10]
Source
Addgene
Catalog Number
213670
Species
Rabbit
Gene / Target
Itga4
Concentration
5 µg/mL
Time
30 min
Temperature
On ice
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
Species
Goat
Concentration
1:200
Time
30 min
Temperature
4 ˚C

Additional Information

Additional Information

Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Mouse 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 B7H3 together with the BFP plasmid. Two days after transfection, cells at a density of 3 x 106 cells/mL were labeled with primary antibodies.

Primary antibody: 5 µg/mL in 1 mL Dulbecco's phosphate-buffered saline (DPBS), 0.5% BSA, with 0.9 mM Ca2+, 0.5 mM Mg2+, kept at 4 °C. 50 µL of antibody added to cells and incubated on ice for 30 min.

Washing conditions: Cells pelleted and washed twice in 0.5% DPBS, 0.5% BSA, with 0.9 mM Ca2+, 0.5 mM Mg2+.

Secondary antibody: 1:200 Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific (Jackson ImmunoResearch, 111-606-046) in 0.5% DPBS, 0.5% BSA, with 0.9 mM Ca2+, 0.5 mM Mg2+, 30 min, 4 °C.

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)