Heparinized peripheral whole blood samples were layered over Ficoll-Hypaque -1077 (Sigma, #H8889) and centrifuged at 400g for 30 minutes after which mononuclear cells (peripheral blood mononuclear cells, PBMNCs) were isolated, washed in saline and counted for subsequent assays. Monocytes were further isolated from mononuclear cells by CD14+ bead selection (Miltenyi Biotech # 130-050-201), counted and checked for purity (92–99%).
Cd14 bead selection
Manufactured by Miltenyi Biotec
The CD14+ bead selection is a lab equipment product used for the isolation and enrichment of CD14+ monocytes from human peripheral blood mononuclear cells (PBMCs). It utilizes magnetic beads coated with anti-CD14 antibodies to selectively bind and separate the CD14+ cells from the PBMC sample.
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Lab products found in correlation
2 protocols using cd14 bead selection
1
Isolation and Purification of Human Monocytes
2
Isolation and Purification of Human Monocytes
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Written consent was obtained by all subjects following a protocol approved by the Dartmouth College Committee for Protection of Human Subjects (institutional review board). Subjects were healthy males and females, 22-50 years of age, taking no medications and with no history of infection or major injury within 90 days. Heparinized whole blood samples were layered over Ficoll-Hypaque-1.077 (Sigma, St Louis, MO, #H8889) and centrifuged at 400g for 30 minutes. Monocytes were isolated from mononuclear cell fractions by CD14+ bead selection (Miltenyi Biotech, Auburn, CA, #130-050-201). Alternatively, mononuclear cells were obtained by leukapheresis and purified using cold aggregation as described. 13 Cells obtained by leukapheresis were stored under liquid nitrogen until use, at which time they were thawed at 37°C and washed twice with RPMI/10% fetal bovine serum (Hyclone, Logan, UT, #SH30070) just before culture. Monocytes obtained by both methods were >92% pure, >90% viable, and gave comparable results.
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