COMBI IC Reagent: Mouse anti CD45 (FITC) and Mouse anti CD14 (PE)
Catalogue number: GCT-201
|Clone||VIT200 and MEM18 |
|Isotype||IgG2a and IgG1 |
|Product Type ||Primary Antibodies|
|Units||50 Tests |
|Species reactivity ||Human|
|Application ||Direct Immunofluorescence|
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
PBS pH 7.2, 1% BSA, 0.05% NaN3
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 µl of EDTA anti-coagulated blood to a 3-5 ml tube
- Add 20 µl 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 µl 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 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
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.
Nordic-MUbio monoclonal antibody reagents contain optimal concentrations of affinity-purified antibody. For stability reasons this monoclonal antibody solution contains sodium azide. These reagents should be stored at 2-8°C (DO NOT FREEZE!) and protected from prolonged exposure to light. If a slight precipitation occurs upon storage, this should be removed by centrifugation. It will not affect the performance or the concentration of the product. 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.
1. Battifora, H. & Trowbridge, I. S. (1983) Cancer 51, 816-21.
Beutler, B. (2002) Curr Top Microbiol Immunol 270, 109-20.
3. Brocklebank, A. M. & Sparrow, R. L. (2001) Cytometry 46, 254-61.
Cobbold, S., Hale, G. & Waldmann, H. (1987) Leucocyte Typing III. p788-803 (Oxford University Press, Oxford-New York-Tokyo).
5. Dalchau, R., Kirkley, J. & Fabre, J. W. (1980) Eur J Immunol 10, 737-44.
6. Goyert, S. M. (1989) Leucocyte Typing IV. p789-793 (Oxford University Press, Oxford-New York-Tokyo).
Goyert, S. M., Ferrero, E., Rettig, W. J., Yenamandra, A. K., Obata, F. & Le Beau, M. M. (1988) Science 239, 497-500.
Goyert, S. M., Ferrero, E. M., Seremetis, S. V., Winchester, R. J., Silver, J. & Mattison, A. C. (1986) J Immunol 137, 3909-14.
Jing, S., Ralph, S., Head, M. T. A., Chain, A. & Trowbridge, I. (1987) Structural studies of the transferrin receptor and T 200 glycoprotein (CD45). Leucocyte Typing III. p899-905 (Oxford University Press, Oxford-New York-Tokyo).
10. Knapp, W. (1982) Blut 45, 301-8.
11. Knapp, W. (1989) Leucocyte Typing IV. p747-780 (Oxford University Press, Oxford-New York-Tokyo).
Means, T. K., Lien, E., Yoshimura, A., Wang, S., Golenbock, D. T. & Fenton, M. J. (1999) J Immunol 163, 6748-55.
13. Nicholson, J. K., Hubbard, M. & Jones, B. M. (1996) Cytometry 26, 16-21.
14. Sugita, K., Majdic, O., Stockinger, H., Holter, W., Burger, R. & Knapp, W. (1987) Transplantation 43, 570-4.
15. Sun, T., Sangaline, R., Ryder, J., Gibbens, K., Rollo, C., Stewart, S. & Rajagopalan, C. (1997) Am J Clin Pathol 108, 152-7.
Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J. & Tobias, P. S. (2000) J Immunol 165, 5780-7.
Thomas, M. L. (1989) Annu Rev Immunol 7, 339-69.
Ugolini, V., Nunez, G., Smith, R. G., Stastny, P. & Capra, J. D. (1980) Proc Natl Acad Sci U S A 77, 6764-8.
19. Yoshimura, A., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R. & Golenbock, D. (1999) J Immunol 163, 1-5.
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