Flow cytometry (see protocol). Immunoprecipitation. Immunohistochemistry on frozen sections. Functional assays. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Protocol
FLOW CYTOMETRY ANALYSIS: Method: 1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cellpopulation with cell separation medium. 2. Wash 2 times. 3. Resuspend the cells to a concentration of 2x10e7 cells/ml in media A. Add 50 µl of thissuspension to each tube (each tube will then contain 1x10e6 cells, representing 1 test). 4. To each tube, add 0. 5-1. 0 µg* of ABIN114228. 5. Vortex the tubes to ensure thorough mixing of antibody and cells. 6. Incubate the tubes for 30 minutes at 4°C. 7. Wash 2 times at 4°C. 8. Add 100 µl of secondary antibody (FITC Goat anti-rat IgG (H+L)) at a 1/500 dilution. 9. Incubate the tubes at 4°C for 30-60 minutes. (It is recommended that the tubes are protected from light since most fluorochromes arelight sensitive). 10. Wash 2 times at 4°C in media B. 11. Resuspend the cell pellet in 50 µl ice cold media B. 12. Transfer to suitable tubes for flow cytometric analysis containing 15 µl of propidiumiodide at 0. 5 mg/ml in PBS. This stains dead cells by intercalating in DNA. Media: A. Phosphate buffered saline (pH 7. 2) + 5% normal serum of host species + sodium azide(100 µl of 2M sodium azide in 100 mls). B. Phosphate buffered saline (pH 7. 2) + 0. 5% Bovine serum albumin + sodium azide (100µl of 2M sodium azide in 100 mls).
Restrictions
For Research Use only
Concentration
1 mg/mL
Buffer
PBS buffer with 0.02 % sodium azide as preservative
Preservative
Sodium azide
Precaution of Use
This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
Avoid repeated freezing and thawing.
Storage
4 °C/-20 °C
Storage Comment
Store the antibody at 2-8 °C for one month or (in aliquots) at -20 °C for longer.
Alpha 4 integrin, which helps to mediate cell-cell and cell-matrix interactions. It combines with beta 1 and beta 7integrin to form VLA-4 and LPAM-1 (Peyers patch homing receptor) respectively. VLA-4 is expressed on most peripheral lymphocytes, thymocytes and monocytes. LPAM-1 is found on peripheral lymphocytes, but few thymocytes. Fibronectin and VCAM-1 act as ligands for both VLA-4 and LPAM-1. LPAM-1 also binds the mucosal vascular addressin MAdCAM-1. (1)Synonyms: CD49 antigen-like family member D, Integrin alpha-4, Integrin alpha-IV, VLA-4, VLA4