DID YOU KNOW? DTIC has over 3.5 million final reports on DoD funded research, development, test, and evaluation activities available to our registered users. Click HERE
to register or log in.
In Vitro Studies of Cryopreserved Granulocytes.
Technical rept. no. 5 (Annual), 10 Dec 76-30 Nov 77,
CENTER FOR BLOOD RESEARCH BOSTON MASS
Pagination or Media Count:
Granulocytes, isolated from the buffy coat of baboon blood by counterflow centrifugation, were frozen in polypropylene tubes in 2.0 ml volumes containing 1 x 10 to the 7th power granulocytes. The medium consisted of 5 DMSO, 6 HES, 4 human serum albumin, and 6 mM glucose in Normosol-R, pH 7.1. The best cooling rate for granulocytes was found to be 4 C for 30 minutes, then 4 C per minute to -80 C and stored for 1-3 weeks in liquid nitrogen at -197 C. Tubes were thawed manually with swirling for 130 seconds at 42 C in a water bath. The yield after thawing was 98 or - 14. Granulocytes isolated from fresh blood all fluoresced green when incubated with fluorescein diacetate FDA. Less than 1 of cells showed nuclear uptake of ethidium bromide EB. After freezing and thawing, an average of 79 or - 8 granulocytes fluoresced green with FDA and 21 or - 8 fluoresced red with EB. About 98 or - 2 of the granulocytes isolated from fresh blood exhibited a serum dependent capacity for ingestion of latex and yeast. Thawed granulocytes were diluted 14 dropwise with a medium containing 7 HES, 4 albumin, and 6 mM glucose in Normosol-R, incubated with fluorescent latex or fluorescent zymosan at 37 C for 30 minutes and then washed twice. After this, 75 or - 9 of the granulocytes fluoresced green with FDA, 76 or - 5 demonstrated serum-dependent latex ingestion and 67 or - 8 ingested zymosan.
APPROVED FOR PUBLIC RELEASE