Cells were thawed as previously described21 (link). Cells were centrifuged and stained with GhostDye510 (Tonbo Biosciences), fixed with 4% formaldehyde, and washed with wash buffer (2% FBS in DPBS). Cells were stained with primary antibodies in 1× BD Perm/Wash (BD Biosciences) +0.2% Triton X-100 (except for Map2 stain, which did not contain Triton X-100) at 4 °C (see Supplementary Table 2 for list of antibodies and dilutions), and labeled with secondary antibodies (where applicable) at room temp. Flow cytometry was performed on a MACSQuant® Analyzer 10 flow cytometer (Miltenyi Biotec). Three biological replicates were analyzed for each maturation time point. Gating strategies are depicted in Supplementary Fig. 4 .
Ghost dye 510
Manufactured by Cytek Biosciences
Ghost Dye 510 is a nucleic acid dye that can be used for cell viability and cell cycle analysis. It binds to DNA and emits fluorescence in the FITC channel.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash, by BD (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Neutrophil monocyte respiratory burst assay kit, by Cayman Chemical (1 mentions)
Cd45 vf450, by Cytek Biosciences (1 mentions)
Cd11b apc cy7, by Cytek Biosciences (1 mentions)
Il 10 jes5 16e3, by BioLegend (1 mentions)
Cd44 im7, by Thermo Fisher Scientific (1 mentions)
Cd19 6d5, by BioLegend (1 mentions)
Pd 1 rmp1 30, by BioLegend (1 mentions)
Cxcr5 2g8, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facs fortessa, by BD (1 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
4 protocols using ghost dye 510
1
Flow Cytometry Analysis of Maturation in Stem Cells
2
Profiling Immune Cell Functionality
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Single-cell brain and bone marrow suspensions were washed twice in 1X sterile, cold PBS, followed by viability staining with Ghost Dye 510 (Tonbo Biosciences) for 30 minutes in the dark at room temperature. Cells were centrifuged at 500g for 5 minutes at 4°C, the supernatant was discarded and the pelleted cells were re-suspended in 100ul of 1:100 FC Receptor Block (Tonbo Biosciences) for 10 minutes at room temperature. Cells were then stained with following surface antibodies: CD45-vf450, CD11b-APC-Cy7, Ly6G-Pe-Cy7, Ly6C-APC (Tonbo Biosciences) for 30 minutes at room temperature, protected from light. Following surface staining, samples were incubated with dihydrorhodamine 1,2,3 (DHR) and 200 nM PMA for 45 minutes at 37°C according to the manufacturer’s instructions (Neutrophil Monocyte Respiratory Burst Assay Kit, Cayman Chemical), then washed and analyzed immediately for rhodamine fluorescence on a Beckmann Coulter Cytoflex S Flow Cytometer. Data was analyzed using FlowJo (TreeStar).
3
Comprehensive Tfh Cell Analysis from Lymph Nodes
4
Single-cell RNA-seq Analysis of Immune Cells
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Cells were stained using anti-human CD45 PE (clone H130, Biolegend) and viability dye (Ghost Dye 510, Tonbo). Viable CD45 + cells were sorted on a Mo o Astrios EQ by the ow cytometry core at the University of Colorado Cancer Center. Target cell number was 5,000 to 10,000 cells. Cells were submitted to the Genomics Core for sequencing using the 10x Platform (10x genomics). Recovered cell number and sequencing depth for each sample is re ected in Supp. Table 2. Sequence data was then analyzed as follows: Cellranger (2.0.2) count module was used for alignment, ltering, barcode counting and UMI counting of the single cell FASTQs. 2D UMAP plots and clustering were determined by the following method: Seurat (V3) was used to lter cells to include only those with > 200 and < 4000 genes, mitochondrial gene expression < 10%, and total counts < 20000. The Seurat module IntergrateData with dims = 1:20 was used to integrate the blood and colon SCRNAseq data from all 8 samples. FindClusters with resolution = 0.25 on PCA reduced data using 30 princIPAl components was used to de ne clusters.
UMAP coordinates were determined by Seurat. Pathway enrichment scores were generated through the use of IPA (QIAGEN Inc.).
UMAP coordinates were determined by Seurat. Pathway enrichment scores were generated through the use of IPA (QIAGEN Inc.).
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