CMFDA Labeling of Platelet

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.

SOLUTIONS

BSGC

IMDM (Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F

ADDITIONAL INFO

We use CMFDA special pack from Probes .

[ read more ] about CMFDA dye.

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.