anti-CD45 (FITC) and anti-CD14 (PE) antibody pair

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Antigen
Reactivity
Human
Host
Mouse
Clonality (Clone)
Monoclonal ()
Conjugate
FITC,PE
Application
Direct Fluorescent Assay (DFA), Flow Cytometry (FACS)
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Supplier Product No.
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Purpose This product is optimised for use with FIX&PERM®.
Brand FIX&PERM®
Clone VIT200-MEM18
Isotype IgG2a, IgG1
Specificity The CD45 mAb (clone VIT200) recognizes a pan CD45 epitope. The CD14 mAb (clone MEM18) recognizes surface CD14 on human monocytes and macrophages as well as neutrophils. The sensitivity of CD45/CD14 mAb is determined by staining well-defined blood samples from representative donors with serial-fold mAb dilutions to obtain a titration curve that allows relating the mAb concentration to the percentage of stained cells and geometric MFI (mean fluorescence intensity). For this purpose, a mAb-concentration range is selected to include both the saturation point (i.e. the mAb dilution expected to bind all epitopes on the target cell) and the detection threshold (i.e. the mAb dilution expected to represent the least amount of mAb needed to detect an identical percentage of cells). In practice, 50 µL of leukocytes containing 10^7 cells/mL are stained with 20 µL mAb of various dilutions to obtain a titration curve and to identify the saturation point and detection threshold. The final concentration of the product is then adjusted to be at least 3-fold above the detection threshold. In addition and to control lot-to-lot variation, the given lot is compared and adjusted to fluorescence standards with defined intensity.
Characteristics Mildly fixes cells, preserving their flow cytometric scatter characteristics
Allows simultaneous characterisation of both intracellular and cell surface markers
Rapid technique - whole procedure can be carried out in less than one hour, ready
for immediate analysis or storage for 24 hours
Stringent QC procedures - the quality of each lot is determined using well-defined
blood samples and subsequent comparison of scatter characteristics of obtained
leukocyte populations, ensuring consistent and reliable results lot after lot
A range of intracellular antibodies with optimised protocols for use with FIX&PERM®
FIX&PERM® is a simple procedure making use of two reagents. Reagent A gently fixes cells, while Reagent B permeabilizes them. The specific formulations reduce background and allow simultaneous addition of permeabilization medium and fluorochrome labelled antibodies, allowing staining of intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins, etc.
Purification Purified by Affinity Chromatography
Components COMBI IC Reagent: Mouse anti CD45 (FITC) and Mouse anti CD14 (PE)
Sub Type Cocktail
Application Notes Staining Procedure Direct Immunofluorescence (Staining Procedure) fluorochrome labeled antibodies are designed for use with either whole blood or isolated mononuclear cell (MNC) preparations. Proposed staining procedure for whole blood in short: - For each sample add 50 µL of EDTA anti-coagulated blood to a 3-5 mL tube - Add 20 µL of the appropriate monoclonal antibody conjugate - Incubate the tube for 15 minutes at 4°C or at room temperature in the dark - Add 100 µL NM-LYSE (ABIN1741580) to each tube and incubate for 10 minutes at room temperature - Add 3-4 mL of destilled water and vortex, incubate for 5-10 minutes at room temperature - Centrifuge tube for 5 minutes at 300 g - Aspirate supernatant and resuspend pellet in 0.3 mL of sheath fluid - Analyze immediately or store samples at 2-8° C in the dark and analyze within 24 hours For \No-Wash\ Proposed staining procedure for MNC in short: - Carefully add 20 µL antibody conjugate and 50-100 µL MNC to the bottom of a tube - Vortex at low speed for 1-2 seconds - Incubate for 15-30 minutes at 2-8°C or at room temperature - Centrifuge tubes for 5 minutes at 300 g - Remove supernatant, resuspend cells in 2-5 mL of phosphate buffered saline (PBS) and centrifuge cells again for 5 minutes at 300 g - Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 mL 1 % formaldehyde and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours
Comment

The CD45 molecule is typically expressed at high levels on all hematopoietic cells. CD45 is a major component of the glycocalix of these cells and can be expressed in different isoforms. Antibody VIT200 recognizes a pan CD45 epitope, which is expressed on all hematopoietic cells. CD14 is a GPI-anchored molecule expressed by virtually all human monocytes and macrophages and - to a lesser degree - granulocytes. CD14 together with Toll-like receptor 4 and MD-2 forms the LPS-receptor complex that recognizes and signals the presence of LPS. While CD14 has no signaling structure its main role seems to be the binding of LPS. The VIT200/MEM18 COMBI-REAGENT-Gate Control permits the identification and enumeration of human leukocytes using flow cytometry. Results must be put within the context of other diagnostic tests as well as the clinical history of the patient by a certified professional before final interpretation. Analyses performed with this antibody should be paralleled by positive and negative controls. If unexpected results are obtained, which cannot be attributed to differences in laboratory procedures, please contact us

Assay Procedure

Procedure: For each sample to be analysed use 50 µL of whole blood, bone marrow or mononuclear cell suspension in a 5 mL tube
Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature)
Incubate for 15 minutes at room temperature
Add 5 mL phosphate buffered saline and centrifuge cells for 5 minutes at 300 g
Remove supernatant and add to cell pellet 100 µL Reagent B (Permeabilization
Medium) and 20 µL of the appropriate monoclonal antibody conjugate
Vortex at low speed for 1-2 seconds
Incubate for 15 minutes at room temperature
Wash cells with phosphate buffered saline as described above
Remove supernatant and re-suspend cells in sheath fluid for immediate analysis or re-suspend cells in 0.5 mL 1.0 % formaldehyde and store at 2-8°C in the dark
Analyse fixed cells within 24 hours

Restrictions For Research Use only
Buffer PBS pH 7.2, 1 % BSA, 0.05 % sodium azide
Preservative Sodium azide
Precaution of Use This reagent contains sodium azide. To avoid the development of hazardous conditions, reagents containing azide should be diluted in running water prior to be discarded. Similar to the work with other biological products, proper handling procedures are recommended.
Handling Advice Do not freeze and protect from prolonged exposure to light.
Storage 4 °C
Storage Comment These reagents should be stored at 2-8°C Stability of the reagent: Please refer to the expiry date printed onto the vial. The use of the reagent after the expiration date is not recommended.
Product cited in: Beutler: "TLR4 as the mammalian endotoxin sensor." in: Current topics in microbiology and immunology, Vol. 270, pp. 109-20, 2002 (PubMed).

Brocklebank, Sparrow: "Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy." in: Cytometry, Vol. 46, Issue 4, pp. 254-61, 2001 (PubMed).

Lünsdorf, Kniep, Kniep: "Immunocytochemical localization of CDw60 antigens on human peripheral T cells." in: Carbohydrate research, Vol. 329, Issue 4, pp. 791-8, 2000 (PubMed).

Tapping, Akashi, Miyake, Godowski, Tobias: "Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 165, Issue 10, pp. 5780-7, 2000 (PubMed).

Yoshimura, Lien, Ingalls, Tuomanen, Dziarski, Golenbock: "Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 163, Issue 1, pp. 1-5, 1999 (PubMed).

Sun, Sangaline, Ryder, Gibbens, Rollo, Stewart, Rajagopalan: "Gating strategy for immunophenotyping of leukemia and lymphoma." in: American journal of clinical pathology, Vol. 108, Issue 2, pp. 152-7, 1997 (PubMed).

Nicholson, Hubbard, Jones: "Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry." in: Cytometry, Vol. 26, Issue 1, pp. 16-21, 1996 (PubMed).

Thomas: "The leukocyte common antigen family." in: Annual review of immunology, Vol. 7, pp. 339-69, 1989 (PubMed).

Goyert, Ferrero, Seremetis, Winchester, Silver, Mattison: "Biochemistry and expression of myelomonocytic antigens." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 137, Issue 12, pp. 3909-14, 1987 (PubMed).

Sugita, Majdic, Stockinger, Holter, Burger, Knapp: "Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens." in: Transplantation, Vol. 43, Issue 4, pp. 570-4, 1987 (PubMed).

Ugolini, Nunez, Smith, Stastny, Capra: "Initial characterization of monoclonal antibodies against human monocytes." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 77, Issue 11, pp. 6764-8, 1981 (PubMed).