Maxpar fix 1 buffer

Immune cells were isolated from the sites of injury in the TBI mice and prepared for cell-staining protocols by Fluidigm. Briefly, cells were stained with 0.5 μM cell-ID cisplatin (Fluidigm, catalog no. 201064) for 2 min, followed by adding 2 ml of MaxPar Cell Staining Buffer (Fluidigm, catalog no. 201068) to stop the reaction. After centrifugation and disposal of the supernatant, the cells were resuspended in MaxPar Cell Staining Buffer to a volume of 50 μl. Fifty microliters of a metal-conjugated surface-marker antibody cocktail was added, and the samples were incubated for 30 min at room temperature. For intracellular staining, cells were fixed in 1 ml of 1× MaxPar Fix I Buffer (Fluidigm, catalog no. 201065) at room temperature for 20 min. After washing twice with 2 ml of MaxPar Perm-S Buffer (Fluidigm, catalog no. 201066), the cells were incubated with 50 μl of an intracellular antibody cocktail for 30 min. After washing twice with 2 ml of MaxPar Cell Staining Buffer, the cells were resuspended in 1 ml of the intercalation solution and incubated overnight at 4°C. After washing twice with 2 ml of MaxPar Cell Staining Buffer, the cell concentration was adjusted to 2.5 to 5 × 10⁵/ml with MaxPar Water (Fluidigm, catalog no. 201069) for the CyTOF assay.

Antibodies were labeled using the X8 antibody labeling kit as per the manufacturer’s protocol (Fluidigm). Single-cell suspensions (1 × 10⁶ cells) were incubated with monoisotopic cisplatin-194Pt (Fluidigm) at RT for 5 min, washed with 1× PBS, incubated with Fc receptor–blocking solution to each tube, and incubated for 10 min at RT, without washing off Fc receptor–blocking solution. The antibody was added in Maxpar Cell Staining Buffer (Fluidigm) at RT for 30 min. After washing with PBS, the cells were fixed with 1× Maxpar Fix I Buffer (Fluidigm) for 30 min at RT, washed twice with Perm-S Buffer (Fluidigm), and then incubated with an intracellular antibody cocktail in Perm-S Buffer for 30 min at RT. The cells were washed and incubated overnight with Cell-ID Intercalator-Ir diluted in Maxpar Fix and Perm Buffer (Fluidigm). After another wash with 0.5% BSA in PBS, the cells were filtered, washed twice with 0.1% BSA, and run in a Helios mass cytometer (Fluidigm) at the University of Pennsylvania. The mass cytometry data were normalized to Equation 4-element calibration bead signal (Fluidigm) in 100-s-interval windows using normalization software version 2 (Fluidigm). Data were analyzed using Cytobank software. A list of reagents is provided in Table S3.

BM cells from two femurs and two tibia were used for mass cytometry obtained by crushing in PBS and immediately fixed with Maxpar Fix I Buffer (Fluidigm) for 30 min at room temperature. For surface staining, cells were incubated with antibodies labeled with Metal-tags (CD150-167ER [cat. no. 3167004B], CD48-156Gd [cat. no. 3156012B], CD117-173Yb [cat. no. 3143001B], SCA-1 169Tm [cat. no. 3169015B], CD41-143Nd [cat. no. 3143009B]; [Fluidigm] and CD105PE [cat. no. 12-1051-82]; [eBioscience], and for the secondary step anti-phycoerythrin [MaxPar Ready] labeled with 160Gd [cat. no. 408105]; [Biolegend]). Next, cells were fixed and stained with specific metal tags antibodies against phosphorylated proteins (pAKT (S473)-152Sm [cat. no. 3152005A], pSTAT5 (Y694) [cat. no. 3150005A], pERK1/2 (T202/Y204)-171Yb [cat. no. 3171010A]; [Fluidigm]). Cells were incubated overnight in MaxPar Fix and Perm Buffer (Fluidigm) with 1:10,000 dilution Cell-ID Intercalator-Ir (Fluidigm), and analyzed on CyTOF2.