CD45 (PTPRC) Mouse Monoclonal Antibody [Clone ID: ML2]

CAT#: AM39019FC-N

CD45 (PTPRC) mouse monoclonal antibody, clone ML2, FITC

Product Images

Representative Data Staining with clone ML2 (anti-CD45) monoclonal antibodies is illustrated by flow cytometry analysis of normal blood cells. Indirect staining was performed using 10 µl of the purified monoclonal antibody with RaM FITC conjugate and 100 µl blood sample.

Specifications

Product Data

• Clone Name: ML2
• Applications: FC
• Recommended Dilution: Clone ML2 can be applied in flow cytometry for analysis of blood and bone marrow samples and in immunohistochemistry using frozen tissue sections or parrafin tissue sections.
  Flow cytometry: please see "Protocols" below.
  Labelled reagent is effectively formulated for direct immunofluorescent staining.
• Reactivities: Human
• Host: Mouse
• Isotype: IgG1
• Clonality: Monoclonal
• Specificity: Anti-CD45 (clone ML2) reacts with the CD45 antigen.
  CD45 clustered antibodies have proven valuable for gate setting of lymphocytes, leukocytes, and monocytes in routine testing using flow cytometry. Combination of CD45 with CD14 antibodies in the analysis of blood (or bone marrow) samples by flow cytometry shows variable expression of these antigens on different cell populations.
  In peripheral blood cells, a distinction can be made between lymphocytes (CD45+++, CD14 -), monocytes (CD45++, CD14++) and granulocytes (CD45++, CD14+/-).
  Testing by flow cytometry using a 'lyse-wash' method on whole blood from healthy donors showed the following values expressed in terms of % of the total lymphocyte count:
  
  Product code: AM39019AC-N (anti-CD45 APC)
  n: 10
  Mean % positive: 99,72
  S.D.: 0,30
  % CV: 0,31
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  Product code: AM39019FC-N (anti-CD45 FITC)
  n: 10
  Mean % positive: 99,43
  S.D.: 0,66
  % CV: 0,66
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  Product code: AM39019RP-N (anti-CD45 PE)
  n: 10
  Mean % positive: 99,75
  S.D.: 0,20
  % CV: 0,21
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• Formulation: 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.3, 0.2% BSA, 0.09% sodium azide
  Label: FITC
  State: Liquid purified Ig fraction
  Label:
  Cat. No. Label EX-max (nm) / EM-max (nm):
  AM39019AC-N APC 595, 633, 635, 647 / 660
  AM39019FC-N FITC 488 / 519
  AM39019RP-N PE 488, 532 / 578
  AM39019PP-N 488, 532 / 678
  AM39019PU-N Pure . /
• Concentration: lot specific
• Purification: Affinity chromatography
• Conjugation: FITC
• Storage: Store the antibody undiluted at 2-8°C.
  Fluorochrome labelled product is photosensitive and should be protected from light.
• Stability: Shelf life: one year from despatch.
• Gene Name: protein tyrosine phosphatase, receptor type C
• Database Link:
  Entrez Gene 5788 Human
  UniProt ID P08575
• Background: The CD45 molecule is also known as the Leukocyte Common Antigen (LCA) or T200 antigen, and is comprised of different glycoproteins ranging from 180-240 kD [1,2]. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Expression of CD45 is found on all hemopoietic cells, e.g. granulocytes, monocytes, macrophages and lymphocytes, except mature erythroid cells. In humans, there is heterogeneous expression of CD45 isoforms (RA, RB, RO, RC) on lymphocyte subpopulations. Detection of the different isoforms can distinguish, for example, between naive T cells and memory T cells, which is of interest in patients with immunodeficiency and autoimmune diseases.
• Synonyms: PTPRC, Leukocyte common antigen, L-CA, T200
• Note: 1. Conjugates with brighter fluorochromes, like PE and APC, will have a greater separation than those with dyes like FITC. When populations overlap, the percentage of positive cells using a selected marker can be affected by the choice of fluorescent label.
  2. Use of monoclonal antibodies in patient treatment can interfere with antigen target recognition by this reagent. This should be taken into account when samples are analyzed from patients treated in this fashion.
  3. Reagent data performance is based on EDTA-treated blood. Reagent performance can be affected by the use of other anticoagulants.
  
  Protocol: Flow cytometry method for use with labelled (FITC, R-PE, APC, PerCP or PerCP-Cy5.5) monoclonal antibodies
  1. Add 100 µl of EDTA-treated blood (i.e. approx. 10e6 leukocytes) to a 5 ml reagent tube. The content of one tube is sufficient to perform one test.
  2. Add to each tube 10 µl of labelled monoclonal antibody. (Appropriate mouse Ig isotype control samples should always be included in any labelling study).
  Vortex the tube to ensure thorough mixing of antibody and cells.
  3. Incubate the tube for 15 minutes at room temperature in the dark.
  4. Add 100 µl of a lyse reagent.
  5. Incubate for 10 minutes at room temperature in the dark.
  6. Add 2 ml of demineralized water and incubate for 10 minutes in the dark.
  7. Centrifuge the labeled cell suspension for 2 minutes at 1000 x g.
  8. Remove the supernatant and resuspend the cells in 200 µl of PBS.
  9. Analyze by flow cytometry within four hours (alternatively, the cells may be fixed by 0.05% of formaline in buffered saline for analysis the next day. Some antigens are readily destroyed upon fixation and this should be taken into account when using this alternative).
  
  Flow cytometry method for use with dual and triple combinations
  1. Add 100 µl of EDTA-treated blood (i.e. approx. 10e6 leukocytes) to a 5 ml reagent tube. The content of one tube is sufficient to perform one test.
  For combinations with anti-kappa and/or anti-lambda Ig see Application note below.
  2. Add to each tube 20 µl of labelled monoclonal antibody combination.
  (Appropriate mouse Ig isotype control samples should always be included in any labelling study).
  3. Vortex the tube to ensure thorough mixing of antibody and cells.
  4. Incubate the tube for 15 minutes at room temperature in the dark.
  5. Add 100 µl of a lyse reagent and mix immediately.
  6. Incubate for 10 minutes at room temperature in the dark.
  7. Add 2 ml of demineralized water and incubate for 10 minutes in the dark.
  8. Centrifuge the labelled cell suspension for 2 minutes at 1000 x g.
  9. Remove the supernatant and resuspend the cells in 200 µl of PBS.
  10. Analyze by flow cytometry within four hours (alternatively, the cells may be fixed by 0.05% of formaline in buffered saline for analysis the next day. Some antigens are readily destroyed upon fixation and this should be taken into account when using this alternative).
  Application note for anti-kappa and/or anti-lambda Ig combinations
  Add 2 ml of PBS containing 0.001% (v/v) Heparin (prewarmed to 37°C) to the cell suspension
  Vortex, centrifuge (2 min at 300x g) and discard the supernatant.
  Repeat this step twice.
  Resuspend the pelleted blood cells in 100 µl PBS, pH 7.2, containing 0.001% (v/v) Heparin.
  
  Flow cytometry method for use with purified monoclonal antibodies
  1. Add 100 µl of EDTA-treated blood (i.e. approx. 10e6 leukocytes) to a 5 ml reagent tube. The content of one tube is sufficient to perform one test.
  2. Add to each tube 10 µl of purified monoclonal antibody. (Appropriate mouse Ig isotype control samples should always be included in any labelling study).
  Vortex the tube to ensure thorough mixing of antibody and cells.
  3. Incubate the tube for 15 minutes at room temperature in the dark.
  4. Wash the labelled cells by adding 2 ml of PBS containing 0.001% (v/v) Heparin, vortexing and centrifuging (2 min 1000 x g) and discard the supernatant.
  5. Add 50 µl of appropriate dilution of F(ab)2 Rabbit Anti Mouse IgG fluorescent conjugate (e.g.FITC or R-PE) in PBS containing 0.001% (v/v) Heparin to the tube. It is recommended that the tube is protected from light.
  6. Mix by vortexing and incubate for 15 minutes at room temperature in the dark.
  7. Add 100 µl of a lyse reagent and mix immediately.
  8. Incubate for 10 minutes at room temperature in the dark.
  9. Add 2 ml of demineralized water and incubate for 10 minutes in the dark.
  10. Centrifuge the labelled cell suspension for 2 minutes at 1000 x g.
  11. Remove the supernatant and resuspend the cells in 200 µl of PBS.
  12. Analyze by flow cytometry within four hours (alternatively, the cells may be fixed by 0.05% of formaline in buffered saline for analysis the next day. Some antigens are readily destroyed upon fixation and this should be taken into account when using this alternative).

Reference Data

• Protein Families: Druggable Genome, ES Cell Differentiation/IPS, Phosphatase, Transmembrane
• Protein Pathways: Cell adhesion molecules (CAMs), Fc gamma R-mediated phagocytosis, Primary immunodeficiency, T cell receptor signaling pathway