Flow Cytometry. Immunoprecipitation. Immunohistochemistry using cryostat sections: (however, cross-reactivity with Lewis ratshave been shown to occur in some instances). Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Protokoll
FLOW CYTOMETRY ANALYSIS: Method: 1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cellpopulation 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 thissuspension to each tube (each tube will then contain 1x10e6 cells, representing 1 test). 4. To each tube, add 0. 5-2. 0 µg of this Ab. 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)) at 1: 500 dilution. Notrecommended for use with PE secondary. 9. Incubate the tubes at 4°C for 30-60 minutes. (It is recommended that the tubes areprotected 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 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). Results - Tissue Distribution: Rat Strain: Brown NorwayCell Concentration: 1x10e6 cells per testAntibody Concentration Used: 0. 5 µg/10e6 cellsIsotypic Control: Mouse IgG2aCell Source Percentage of cells stained above control: Thymus 39. 8%Spleen 95. 9%Lymph Node 99. 9%Results - Strain Distribution: Cell Concentration: 1x10e6 cells per testAntibody Concentration Used: 0. 5 µg/10e6 cellsStrains Tested: Lewis, Wistar, Brown Norway, Fischer 344, Buffalo, ACIPositive: Brown NorwayNegative: Lewis, Wistar, Fischer 344 Buffalo, ACI
Beschränkungen
Nur für Forschungszwecke einsetzbar
Konzentration
1.0 mg/mL
Buffer
PBS and 0.02 % NaN3
Konservierungsmittel
Sodium azide
Vorsichtsmaßnahmen
This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handhabung
Avoid repeated freezing and thawing.
Lagerung
4 °C/-20 °C
Informationen zur Lagerung
Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.
MHC Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen. MHC class I antigens are heterodimers consisting of one alpha chain (44 kDa) with beta 2 microglobulin (11.5 kDa). The antigen is expressed by all somatic cells at varying levels. MHC Class I molecules are expressed on most nucleated cells where they present endogenously synthesized antigenic peptides to CD8+ T lymphocytes, which are usually cytotoxic T cells. Fibroblasts or neurons however only show a low level of antigen.Synonyms: MHC class I RT1.Ac heavy chain, RT1-A3