Flow cytometry for Anti-Integrin alpha 5 [IPI-MFR5]
Addgene #213671
- 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 5 [IPI-MFR5] (Addgene #213671) in a flow cytometry assay with a mouse sample.
![Histogram from FACS analysis on ExpiCHO cells transfected with mouse integrin α5β1 (red) or B7H3 (gray). Cells were labeled with Anti-Integrin alpha 5 [IPI-MFR5] (Addgene #213671) 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.](https://datahub-media.addgene.org/report-images/c5xh6m/Flow-213671-Anti-Integrin_alpha_5_IPI-MFR5.png)
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 [IPI-MFR5]
Addgene #213671 -
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 α5β1.
Primary Antibody
- Name
- Anti-Integrin alpha 5 [IPI-MFR5]
- Source
- Addgene
- Catalog Number
- 213671
- Species
- Rabbit
- Gene / Target
- Itga5
- Concentration
- 5 µg/mL
- Time
- 30 min
- Temperature
- On ice
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)