Anti-Integrin alpha V beta 5 [IPI-ITGAV/ITGB5.9] in Flow Cytometry (Human)
Addgene #213659
- 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 5 [IPI-ITGAV/ITGB5.9] (Addgene Antibody 213659) in a flow cytometry assay with a human sample.
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 5 [IPI-ITGAV/ITGB5.9]
Addgene #213659 -
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 5 [IPI-ITGAV/ITGB5.9]
- Source
- Addgene
- Catalog Number
- 213659
- Species
- Rabbit
- Gene / Target
- ITGAV, ITGB5
- Concentration
- 50 nM
- Time
- 60 min
- Temperature
- 4 ˚C
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)