CD65s antibody

Catalog No. ABIN1741612

This Mouse Monoclonal antibody specifically detects in FACS, DFA and IFA. It exhibits reactivity toward Humanand has been mentioned in 1 publication.

Quick Overview for CD65s antibody (ABIN1741612)

Target: CD65s

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: Un-conjugated

Application: Flow Cytometry (FACS), Direct Fluorescent Assay (DFA), Indirect Immunofluorescence Assay (IFA)

Clone: VIM2

Product Details

Purpose: This product is optimised for use with FIX&PERM®.

Brand: FIX&PERM®

Specificity: Antibody VIM2 reacts with a carbohydrate structure expressed by myeloid cells. The epitope recognized was shown by Macher et al. to involve a defined sialofucooligosaccharide sequence. Similar results were obtained by Kniep et al.. Together with other sialylated and fucosylated polylactosamines the carbohydrate structure recognized by VIM2 may play a critical role on the adhesion of granulocytes and monocytes to endothelium and platelets during inflammation and clotting. The sensitivity of VIM2 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 fluorescenceintensity). 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, 50ul of leukocytes containing 10^7cells/mL are stained with 20ul 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 three-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

Isotype: IgM

Application Details

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 Indirect Immunofluorescence (Staining Procedure) - Mix 20 µL purified antibody with 50 µL whole blood or MNC suspension - Incubate for 15 minutes at 2-8°C - Wash cells with phosphate buffered saline (PBS) - Add to cell pellet 20 µL of affinity purified, fluorochrome labeled F(ab')2 anti mouse Ig antibodies - Incubate for 15 minutes at 2-8°C - Wash cells with phosphate buffered saline (PBS) or proceed as described for direct staining

Comment:

The epitope recognized by antibody VIM2 is expressed by virtually all myeloid cells including normal and malignant granulocytes and monocytes. In normal myelopoiesis VIM2 can first be detected after the late CFU-GM stage. In acute myeloid leukemias (AMLs) in vitro clonogenic progenitors seem to aberrantly express the VIM2 antigen. A variety of studies have demonstrated the usefulness and reliability of VIM2 as a marker molecule for the classification of acute leukemias. Recently, the signal transducing capacity of VIM2 bearing surface molecules has been demonstrated. The VIM2 antibody permits the identification and enumeration of normal and leukemic cell populations expressing the VIM2 antigen present in human biological samples (blood, bone marrow and others) using flow cytometry. Furthermore, VIM2 mAb is suitable for the elimination of myeloid cells from complex cell mixtures as well as for functional studies. (Lund-Johansen et al.) 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.

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

Handling

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.

References

Bettelheim, Panzer, Majdic, Stockinger, Roithner, Köller, Meryn, Lechner, Knapp: "Unexpected absence of a myeloid surface antigen (3-fucosyl-N-acetyllactosamine) in promyelocytic leukemia." in: Leukemia research, Vol. 9, Issue 11, pp. 1323-7, (1986) (PubMed).

Target Details

Target: CD65s