CMFDA (5-chloromethylfluorescein diacetate) is a lipophilic tracer that has an enormous advantage over ordinary tracers (e.g. FITC) because it can be introduced into live (!) cells. Once inside the cell this dye undergo some covalent changes restricting from its passgae to extracellular milieu.
PROTOCOL
- Collect blood and purify platelets.
- Resuspend in the certain known volume of BSGC and count platelets. Separate 10x10^6 platelets (negative control). Centrifuge at 3000 rpm at 4ѓC, for 7 min. Store them resuspended in Iscove's Modified Dulbecco's Medium (IMDM), at 4ѓC before the FACS preparation starts.
- Resuspend 3.2x10^8 platelets in 5 ml of BSGC together with 50µg of CMFDA in 15 ml Falcon Tube. Store for 60 min., at RT, at dark with slight rotation.
- Centrifuge at 3000 rpm at 4ѓC, for 7 min.
- Resuspend in 5 ml of BSGC.
- Centrifuge at 3000 rpm, at 4ѓC, for 7 min.
- Let to stay for 60 min., at RT, at dark (elimination of CMFDA access)
- Centrifuge at 3000 rpm, at 4ѓC, for 7 min.
- Resuspend in the certain known volume of BSGC and count platelets. Separate 10x10^6 platelets (quality control of labeling). Centrifuge at 3000 rpm at 4ѓC, for 7 min. Store them resuspended in IMDM, at 4ѓC before the FACS preparation starts.
- The main part of the labeled platelets should be centrifuged, resuspended in the respective solution (NaCl 0.9% for in vivo use, IMDM, HCF or PBS for in vitro use), counted, and utilized in subsequent in vitro or in vivo experiments.
- BSGC
- IMDM (Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F
SCHEME
REFERENCES
- Baker, G.R. and Levin, J. (1998). Transient thrombocytopenia produced by administration of macrophage colony-stimulating factor: investigations of the mechanism. Blood 91(1): 89-99.
- Levin, J., Peng, J.P., Baker, G.R., Villeval, J.L., Lecine, P., Burstein, S.A. and Shivdasani, R.A. (1999). Pathophysiology of thrombocytopenia and anemia in mice lacking transcription factor NF-E2. Blood 94(9): 3037-47.
