Mouse anti CD65s

Staining Procedure
Direct Immunofluorescence (Staining Procedure)
Nordic-MUbio 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 ?l of EDTA anti-coagulated blood to a 3-5 ml tube
- Add 20 ?l of the appropriate Nordic-MUbio monoclonal antibody conjugate
- Incubate the tube for 15 minutes at 4°C or at room temperature in the dark
- Add 100 ?l NM-LYSE (Cat.No. GAS-003) 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? protocol please refer to www.nordicmubio.com

Proposed staining procedure for MNC in short:
- Carefully add 20 ?l antibody conjugate and 50-100 ?l 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 ?l Nordic-MUbio purified antibody with 50 ?l 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 ?l 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

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. If unexpected results are obtained which cannot be attributed to differences in laboratory procedures, please contact us.

For professional users only.
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.

PBS pH 7.2, 1% BSA, 0.05% NaN3

PBS pH 7.2, 1% BSA, 0.05% NaN3

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 10^7
cells/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.