Legendplex bead array

A 13-plex nonhuman primate specific cytokine/chemokine LEGENDplex bead array (BioLegend) was used according to the manufacturer protocol on plasma and BAL fluid samples. All samples were run on a BD FACS Aria and analysis was performed using LEGENDplex analysis software (Biolegend). An ELISA specific for cynomolgus/rhesus IFN-α2 was performed using BAL samples according to manufacturer protocol (PBL, VeriKine). BAL fluids from post-infection time points were diluted 50-fold in order to remain within the standard limits of detection. An anti-monkey albumin ELISA was used to quantify albumin concentrations in the BAL fluid and plasma (Innovative Research). Plasma was diluted according to the manufacturer protocol and BAL fluid was diluted either 1,000-fold for pre-infection samples or 20,000-fold for post-infection samples.

Microglia were isolated from the brain from a naïve male rat (n=1) as previously described using the Neural Tissue Dissociation Kit and GentleMACS Dissociator (Miltenyi Biotec).¹⁰ Using Percoll centrifugation, myelin was removed from the cells. The brain cells were then cultured as previously described.¹⁰ First, we coated a 48-well plated with cell matrix basement membrane gel (ATCC, Manassas, VA). We then plated 2.5 ×10⁵ cells/well in microglia-specific media (DMEM + 10% FBS), and media was changed every 48 hours. After 6 days, the cells were activated with LPS (1 μg/mL). 2 hours after activated, Treg (1:2 Treg:microglia), MSC (1:10 MSC:microglia) or combination of Treg (1:2) +MSC (1:10) were added to the wells; MSC were added either 1) concurrently with Treg or 2) 18 hours after activation.
The culture supernatant was collected at 72 hours. All samples were analyzed using a 13-plex rat-specific cytokine/chemokine LEGENDplex bead array according to the manufacturer’s protocol (Biolegend, San Diego, CA #740401). The samples were analyzed using a BD LSRII, and analysis was performed using LEGENDplex analysis software (Biolegend).