Immunohistochemistry with frozen sections. This clone has been reported to be unsuitable for use with paraffin sections. Flow cytometry.
Reactivity
Rat
Antibody Host
Mouse
Isotype
IgG2a
Clonality
Monoclonal
Immunogen
Lymph node glycoproteins and cells
Specificity
Antibody CL111A recognizes a monomorphic determinant of the leukocyte common antigen.(1)The antigen recognized is a heavily glycosylated membrane glycoprotein of molecular weight 170,000 kDa on thymocytes but molecular weight 170,000-220,000 kDa on other leukocytes. Results for: Tissue distribution by flow cytometry analysis (Rat Strain: Wistar): Cell source thymus: Percentage of cells stained above control = 99.9% Cell source spleen: Percentage of cells stained above control = 97.4% Cell source lymph node: Percentage of cells stained above control = 90.6% (Cell concentration = 1x10e6 cells per test, antibody concentration used = 0.5 μg/10e6 cells, isotypic control Mouse IgG2a). Strain distribution by flow cytometry analysis: Procedure: see below. Antibody concentration used = 0.5 μg/10e6 cells Strains tested: Wistar, Buffalo, Brown Norway, Fischer 344 Positive: Wistar, Buffalo, Brown Norway, Fischer 344 Negative: none
Buffer
State: Azide Free State: Liquid Ig fraction in PBS
Concentration
lot specific
Purification
Protein G affinity chromatography
Conjugation
Unconjugated
Storage
Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing.
The leukocyte common antigen (L-CA) is a major glycoprotein of haematopoietic cells but is not found on other tissues or erythroid cells. It is present on greater than 95% of thymocytes, bone marrow cells and thoracic duct lymphocytes. This molecule carries much of the carbohydrate of thymocytes and shows interesting heterogeneity amongst T lymphocytes and B lymphocytes. (2,3)
Synonyms
PTPRC, Leukocyte common antigen, L-CA, T200
Note
Protocol: FLOW CYTOMETRY ANALYSIS: 1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population 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 contain 1 x 10e6 cells, representing 1 test). 4. To each tube, add 0.5-1.0 µg of CL111A. 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-mouse IgG (H+L)). 9. Incubate the 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 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 (100 µl of 2M sodium azide in 100 mls).
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