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.
Maxpar perm s buffer
Manufactured by Standard BioTools
Sourced in United States
Maxpar Perm-S Buffer is a permeabilization buffer designed for use with flow cytometry. It is used to prepare single-cell suspensions for intracellular staining and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Maxpar cell staining buffer, by Standard BioTools (2 mentions)
Maxpar fix 1 buffer, by Standard BioTools (2 mentions)
Cell id cisplatin, by Standard BioTools (1 mentions)
Maxpar water, by Standard BioTools (1 mentions)
Helios mass cytometry platform, by Standard BioTools (1 mentions)
Cell acquisition solution, by Standard BioTools (1 mentions)
Cell id iridium intercalator solution, by Standard BioTools (1 mentions)
3 protocols using maxpar perm s buffer
1
Immune Cell Profiling of TBI Mice
2
Comprehensive Cytoplasmic Antigen Staining
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Cells were stained with surface and intracellular markers following the Maxpar Cytoplasmic/Secreted Antigen Staining with Fresh Fix Protocol (Fluidigm, USA). Briefly, cells were washed with PBS to remove excess Ag NPs and then stained with cisplatin for viability [31 (link)]. Subsequently, cells were stained with the surface markers listed in Table S2 . After surface staining, cells were fixed in Maxpar Fix I Buffer (Fluidigm, South San Francisco, CA, USA) and permeabilized with Maxpar Perm-S Buffer (Fluidigm, CA, USA). Then, cells were stained with intracellular markers (Table S2 ). After intracellular staining, cells were fixed again with 1.6% formaldehyde and stained with Cell-ID Intercalator-Ir. Prior to data acquisition, cells were washed and suspended at 1 × 106 cells/mL in Cell Acquisition Solution (Fluidigm, CA, USA). Calibration beads were added 1:10 by volume for normalization. Cells were then filtered into strainer-capped tubes and samples were analyzed on the Helios mass cytometry platform (Fluidigm, CA, USA).
3
Comprehensive Cell Immunophenotyping Protocol
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Extracellular staining was performed on pooled barcoded cells in Maxpar Cell Staining Buffer (Fluidigm) for 30 minutes at room temperature. Intracellular staining (perforin) was performed in Maxpar Perm-S Buffer (Fluidigm) for 30 minutes at room temperature. Stained cells were then incubated for 10 minutes in 1.6% formaldehyde (FA) freshly prepared from 16% stock FA (Sigma-Aldrich). DNA staining was performed by overnight incubation at 4°C in 2mL of 125nM Cell-ID Iridium intercalator solution (Fluidigm). Cells were then washed, pelleted, and kept at 4°C until acquisition the same day.
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