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Accession Number:
ADA048661
Title:
In Vitro Studies of Cryopreserved Granulocytes.
Descriptive Note:
Technical rept. no. 5 (Annual), 10 Dec 76-30 Nov 77,
Corporate Author:
CENTER FOR BLOOD RESEARCH BOSTON MASS
Report Date:
1977-12-15
Pagination or Media Count:
29.0
Abstract:
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.
Distribution Statement:
APPROVED FOR PUBLIC RELEASE
