Anti cd44 fitc

Cell cycle analysis was carried out using propidium iodine (PI) staining. In short, after exposure to ⁹⁰SrCl₂, cells were detached by trypsin incubation, washed twice and counted. 300,000 cells were fixed in 70% ethanol and stored at −20 °C for at least one hour. Cells were then centrifuged 8 minutes at 400 g and the pellet was re-suspended with a sodium citrate solution containing 0.2% Triton X-100, 100 μg.mL⁻¹ RNAse and 50 μg.mL⁻¹ PI (Sigma) and incubated 30 minutes at 37 °C in the dark. Stained cells were then analyzed on a FACS Canto II (BDIS, Le Pont-de-Claix, France) with the acquisition of at least 10,000 events per condition.
Phenotypic analysis of rat BMSCs was performed using the following directly coupled antibodies: anti-IgG1-FITC, anti-IgG2a-PE, anti-CD44-FITC, anti-CD45-FITC, anti-CD90-PE, anti-CD73-alexa647 (all from BD Pharmingen, Le Pont-de-Claix, France) and anti-CD34-PE (Santa Cruz Biotechnologies). At the first passage, aliquots of 200,000 cells were washed twice in PBS and incubated for 20 minutes in the presence of the indicated antibodies at pre-defined concentrations. Cells were then washed twice and analyzed.

Groups of mice were sacrificed at the following time points: uninfected (day 0) and post-infection (days 5 and 14). At the indicated time points, spleens were collected from all animals and single cell suspensions were prepared. Cells were stained with anti-CD4-PO (Invitrogen), anti-CD44-FITC and anti-Thy1.1-PerCP (all from BD Pharmingen), anti-CD8-PB, anti-PD-1-FITC, anti-BTLA-PE, and anti-2B4-allophycocyanin (all from eBioscience).
For intracellular cytokine staining, single-cell suspensions of splenocytes were plated in a 96-well plate (1×10⁶ cells per well) in culture medium containing RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 mg/ml gentamicin (Mediatech), and 5×10⁻⁵ M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Cells were incubated for 4 h in 10 nM OVA_257-264 (SIINFEKL; Emory University Microchemial Core Facility) and 10 mg/ml brefeldin A (Pharmingen). Following incubation, cells were stained with anti-CD4-PO (Invitrogen), anti-Thy1.1-PerCP (BD Pharmingen), and anti-CD8-PB (eBioscience) and processed using an intracellular staining kit (BD Biosciences) and stained with anti-IFN-γ-Alexa 700 and anti-IL-2-FITC (BD Biosciences). All samples were run on a LSRII flow cytometer (BD Biosciences), and data was analyzed using FlowJo 9.5 Software (Tree Star, San Carlos, CA).