Fixable viability dyes efluor 506

Intracellular RNA flow cytometry was performed using PrimeFlow RNA (Affymetrix, Santa Clara, CA) reagents following manufacturer’s protocols. Cell viability was assessed by Fixable Viability dyes eFluor 506 or eFluor 455UV (eBioscience). Cell surface markers were stained using antibodies against CD45 (clone 30-F11), CD11b (clone M1/70), Ly6G (clone 1A8), and Ly6C (clone HK1.4). A DapB RNA probe, a probe for RNA of a bacterial gene, served as a control for non-specific nucleic acid binding that could occur in activated macrophages. The following RNA probes were used: DapB, Arg1, Mrc1, Chi3l3, Tnf, and Il1b. Cell staining was analyzed on a FACSAria IIu located at the San Francisco VA Medical Center or the San Francisco General Hospital. Data analysis was performed by using FlowJoX (Treestar, Ashland, OR). For BMDM, three independent RNA flow cytometry experiments were performed. For TBI mice, six separate RNA flow cytometry experiments were performed, each with pooled ipsilateral hemispheres from eight age-matched cage mates. For sham-injured mice, three independent RNA flow cytometry experiments were performed, with pooled ipsilateral hemispheres from six to ten age-matched cage mates.

Mitochondrial volume was measured by loading Jurkat cells with 100 nM Mitotracker red CMXRox (Invitrogen). Cells were harvested with Fixable Viability Dyes eFluor 506 (eBioscience) prior to fixation. Fixed and permeabilized cells were subjected to immunostaining with Alexa Fluor 647 Rat anti-Histone H3 (pS28) (Clone HTA28) antibody, a well-known marker for mitosis (BD Biosciences). The measurement of mitochondrial volume was conducted using a BD Fortessa X20 cell analyzer. Viable naturally occurring mitotic and interphase cells were analyzed. Flow cytometry data were analyzed by the FlowJo software (BD).