Itgam Mouse Monoclonal Antibody [Clone ID: WT.3]

CAT#: SM264B

Itgam mouse monoclonal antibody, clone WT.3, Biotin

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Specifications

Product Data

• Clone Name: WT.3
• Applications: FC, IHC
• Recommended Dilution: Immunoprecipitation.
  Flow cytometry.
  Immunohistochemistry on cryostat sections.
  Functional studies: in vivo/in vitro.
• Reactivities: Rat
• Host: Mouse
• Isotype: IgG1
• Clonality: Monoclonal
• Specificity: This Antibody is specific for the β subunit of LFA-1. This antibody also recognizes MAC-1 and p150, 95 as well as LFA-1, since they all contain the same β subunit. It inhibits homeotypic aggregation of PHA blasts and blocks the binding of rat lymphocytes to purified rat ICAM-1
• Formulation: PBS, pH 7.2, with 0.09% NaN3 and 0.5% BSA.
  Label: Biotin
  State: Liquid purified Ig
• Concentration: 0.1 mg/ml
• Conjugation: Biotin
• Database Link:
  Entrez Gene 25021 Rat
• Background: LFA-1 (lymphocyte function associated molecule-1) is one of the leukocyte integrins. It is a heterodimer consisting of α and β subunits of 160-170 kDa and 95-100 kDa respectively. LFA-1 promotes non-antigen dependent adhesion of T-cells to a variety of lymphoid cells that bear its complementary receptor I-CAM-1 (1). It has a broad distribution and is found on most common lymphocytes.
• Synonyms: Integrin beta-2, MFI7
• Note: Protocol: FLOW CYTOMETRIC ANALYSIS:
  
  Method:
  1. Prepare cell suspension in Media A. For cell preparations, deplete the red blood cell population with Lympholyte®-Rat 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 this suspension to each tube (each tube will then contains 1x10e6 cells. representing 1 test).
  4. To each tube add 1.0 µg of this Ab per 10e6 cells.
  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 detection reagent (Streptavin-FITC) at a 1/700 dilution.
  9. Incubate tubes at 4°C for 30-60 minutes (It is recommended that the tubes are protected from light since most fluorochromes are light 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 propidium iodide at 0.5 mg/ml in phosphate buffered saline. (This stains dead cells by intercalating 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 (100 µl of 2M sodium azide in 100 mls).
  
  Results - Tissue Distribution by Flow Cytometry Analysis:
  (Representative Histogram)
  Rat Strain: Wistar
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration used: 1.0 µg/10e6 cells
  Isotypic Control: Biotin Mouse IgG1,κ
  
  Cell Source Percentage of cells stained above control:
  Thymus: 94.5%