Rectal Mucosal Mononuclear Cell Isolation

Mucosal mononuclear cells (MMC) were isolated from rectal biopsies using a combination of mechanical and enzyme digestion as previously described [10 (link)]. Flow cytometric analysis was performed on a BD LSRFortessa cytometer (BD Biosciences, San Jose, CA). All data were stored in list mode and analyzed with BD FACSDIVA operating system and Flow Jo (Tree Star, Inc., Ashland, OR). All antibodies were purchased from BD Biosciences, San Jose, CA (PerCP-CD45, Clone 2D1; Pacific Blue-CD3, Clone UCHT1; PE-Cy7-CD4, Clone SK3; APC-H7-CD8, Clone SK1; FITC-CD69, Clone FN50; APC-CD184 (CXCR4), Clone 12G5 and PE-CD195 (CCR5)) and titrated under assay conditions to determine an optimum saturating dilution. Cells were stained with LIVE/DEAD Fixable Aqua stain fluorescence (Life Technologies, Eugene, OR) to define viable cells. The gating strategy used in this study can be found in S1 Fig.

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Mcgowan I., Cranston R.D., Duffill K., Siegel A., Engstrom J.C., Nikiforov A., Jacobson C., Rehman K.K., Elliott J., Khanukhova E., Abebe K., Mauck C., Spiegel H.M., Dezzutti C.S., Rohan L.C., Marzinke M.A., Hiruy H., Hendrix C.W., Richardson-Harman N, & Anton P.A. (2015). A Phase 1 Randomized, Open Label, Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Three Formulations of Tenofovir 1% Gel (the CHARM-01 Study). PLoS ONE, 10(5), e0125363.

Publication 2015

BD LSRFortessa cytometer PE-CD195 LIVE/DEAD Fixable Aqua stain fluorescence

Antibodies Assay Biopsies Ccr5 Cells Clone Cxcr4 Digestion Dilution Enzyme Fitc Flow cytometric Fluorescence Mucosal Rectal Stain Tree

Corresponding Organization : Johns Hopkins University

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Variable analysis

independent variables

Mechanical and enzyme digestion to isolate mucosal mononuclear cells (MMC) from rectal biopsies

dependent variables

Flow cytometric analysis of MMC
Expression of CD45, CD3, CD4, CD8, CD69, CXCR4 (CD184), and CCR5 (CD195) on MMC

control variables

LIVE/DEAD Fixable Aqua stain to define viable cells

controls

No positive or negative controls were explicitly mentioned.

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