Mouse anti CD14 conjugated to FITC

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

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 MEM18 antibody permits the identification and enumeration of leukocytes using flow cytometry. MEM18 has been also used for functional studies since this antibody blocks the interaction of LPS with CD14 on monocytes. 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

FITC

2 ml of FITC-conjugated anti CD14 (clone MEM18) in PBS pH 7.2, 1% BSA, and 0.05% NaN3, approximately 100 tests.

FITC

The CD14 mAb (clone MEM18) recognizes surface CD14 on human monocytes and macrophages as well as on neutrophils.

The sensitivity of MEM18 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 ?l of leukocytes containing 10^7 cells/ml are stained with 20 ?l 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.

P08572