CD14 Antibody – Mouse

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 ul of EDTA anti-coagulated blood to a 3-5 ml tube
- Add 20 ul of the appropriate monoclonal antibody conjugate
- Incubate the tube for 15 minutes at 4 degree C or at room temperature in the dark
- Add 100 ul NM-LYSE 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 degree C in the dark and analyze within 24 hours
Proposed staining procedure for MNC in short:
- Carefully add 20 ul antibody conjugate and 50-100 ul MNC to the bottom of a tube
- Vortex at low speed for 1-2 seconds
- Incubate for 15-30 minutes at 2-8 degree 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 degree C in the dark. Analyze
fixed cells within 24 hours

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