Primeflow rna

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.

Intracellular RNA flow cytometry was conducted using the manufacturer's protocol (PrimeFlow RNA, Affymetrix, Santa Clara, CA). RPL13A RNA probe was used as a positive control and a no probe negative control was included. The following Type I RNA probes were obtained from eBioscience: IL21R, SP1, PRDM1, SOCS1, SOCS3, and miR155.

Double in situ hybridization in single cells for RNA flow cytometry was performed using PrimeFlow RNA (Affymetrix, Santa Clara, CA) reagents following the manufacturer’s protocols. Cell viability was assessed by live/dead fixable dead cell, violet (ThermoFischer; L34955). Hsp90ab RNA probe (a gene expressed ubiquitously) served as a positive control. Hoxd11 and Hoxd13 RNA probes were used for the actual analysis. Cell staining was analyzed on a FACS Astrios located at the EPFL flow cytometry platform. Data analysis was performed by using FlowJoX (Treestar, Ashland, OR). The labelling and flow cytometry were performed on dissociated cells from eight forelimbs obtained from four different animals pooled together.