Up to 5 × 106 leukocytes isolated from each sample were used for downstream sorting. Cells were first fixed in 1 mL of Cell Cover, a light preservative that maintains RNA integrity (Anacyte #800–250), on ice for 10 minutes, then centrifuged. CD3+ cells were removed from total cell suspensions by magnetic selection (StemCell #17851) according to manufacturer’s instructions. CD3- fractions were incubated on ice for 12 minutes in PBS supplemented with 0.5% FBS (Gibco #16140–071) using the following antibodies: CD45 (HI30, Tonbo Biosciences #50–0459-T100), CD11b (ICRF44, Tonbo #35–0118-T100), CD14 (M5E2, Biolegend #301820), CD16 (3G8, BD #560474), CD66a/c/e (ASL-32, Biolegend #342310), CD2, (RPA-2.10, Biolegend #300204), CD20 (2H7, Biolegend #302349), CD56 (HCD56, Biolegend #318320), and viability dye (Life Technologies, #L34972). After washing out the antibody stain with HBSS, samples were stained for 20 minutes on ice with viability dye diluted in HBSS (Life Technologies #L34972). Cells were resuspended in PBS plus 0.5% FBS and myeloid populations were sorted using a FACSaria II (Fig. S2 ) directly into RNA lysis buffer composed of 200 μL RA1 buffer (Macherey-Nagel #RA1) freshly spiked with 2% TCEP (Thermofisher #77720).
Viability dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Viability dye is a fluorescent stain used to detect live and dead cells in a sample. It is a core tool for cell analysis and viability studies.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (6 mentions)
Hyaluronidase, by Merck Group (4 mentions)
Fc block, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
Anti cd4, by BD (3 mentions)
Hla dr apc cy7, by BioLegend (3 mentions)
Cd4 bv605, by BD (3 mentions)
Cd11b pe cy5, by BioLegend (3 mentions)
Cd3 pe cy7, by BD (3 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Lsr 2, by BD (3 mentions)
Cytofix fixation buffer, by BD (3 mentions)
Perm wash buffer, by BD (3 mentions)
Annexin 5, by BD (2 mentions)
Dnase 1, by Roche (2 mentions)
Golgiplug, by BD (2 mentions)
Fortessa, by BD (2 mentions)
Foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Cd14 v450, by BD (2 mentions)
78 protocols using viability dye
1
Myeloid Cell Sorting for RNA Analysis
2
CD8+ T Cell-Mediated Cytotoxicity Assay
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Previously cryopreserved CD8+ G6PC2-reactive TCR avatars (clone 32) were thawed and rested for 24 h with the homeostatic cytokine IL-7 (10 ng/ml) prior to coculture with MP-treated HLA-A2+ genotype selected (57 (link), 58 (link)) DCs for 24 h in tissue culture treated plates at a 1:1 DC : CD8+ T cell ratio. Nonadherent CD8+ T cell avatars were removed from the plate, washed, and resuspended in fresh media for the killing assay. The human β-cell line, βlox5 (59 (link), 60 (link)), was maintained under standard culture conditions, labeled with CTV fluorescent dye, and plated 18 h prior to the killing assay to achieve 80-90% confluency. At time of assay, media was removed and CD8+ T cell avatars were seeded at an effector to βlox5 target cell ratio of 5:1. Cell death was assessed at 18 h via flow cytometric analysis of Annexin V (BD Biosciences) and viability dye (Life Technologies) staining via an assay established in the lab (61 (link)).
3
Quantification of Antigen-Specific CD8+ T Cells
4
Isolation of Colonic Epithelial and Lamina Propria Cells
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Colonic epithelial and lamina propria cells from mice were isolated using a modified version of a previously described method [23 (link)]. In brief, colons were collected, cut open longitudinally into 1 cm pieces, and washed in calcium- and magnesium-free HBSS (Gibco) supplemented with 2% heat-inactivated fetal calf serum (FCS, Wisent) and 15 mM HEPES (Gibco). The resulting tissue pieces were washed in calcium- and magnesium-free HBSS supplemented with 2% FCS, 15 mM HEPES, and 5 mM EDTA to remove epithelial cells, which were then collected by centrifugation. After removing the supernatant, the tissue pieces were incubated in RPMI-1640 (Sigma) supplemented with 10% FCS, 15 mM HEPES, 160 μg/ml collagenase IV (Sigma) and 40 μg/ml DNAse I (Roche) for 40 min at 37°C. The cell suspension was filtered through a 70 μm cell strainer (Sigma) before proceeding with antibody staining. Cells were stained with viability dye (Life Technologies) and surface antibody CD45.2 (eBioscience) and sorted on the FACSAria II (BD Biosciences) into CD45+ (hematopoietic) and CD45- (non-hematopoietic) populations. R-spondin expression was assessed by qRT-PCR using Gapdh as the housekeeping gene.
5
Evaluating Long Peptide Immunogenicity
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The immunogenicity the long peptides was evaluated in cryopreserved peripheral blood mononuclear cells (PBMC) from the three subjects as described (74 (link)). PBMC were thawed, rested overnight in RPMI 10% FBS with Penicillin/Streptomycin. For the in vitro stimulation (IVS), cells were plated in 24- to 96-well plates at 2 × 106 cells per well in RPMI, 8% human serum supplemented with Penicillin/Streptomycin, 50 μM beta-mercaptoethanol and recombinant human IL-2 at a final concentration of 100 UI/ml. The cells were stimulated with peptide pools containing 1 μg/ml of each candidate peptide. At day 12, intracellular cytokine stainings (ICS) were performed. Each individual well was splitted in two identical fractions and one fraction only was re-challenged with 1 μg/ml of the corresponding peptide for 16–18 h at 37°C and 5% CO2 in presence of 1 μg of brefeldin A (Golgiplug, BD). As a positive control, cells stimulated with staphylococcal enterotoxin B (SEB) at a concentration of 0.25 ng/ml. After 16–18 h of re-stimulation with individual long peptides, cells were harvested and stained with anti-CD3, anti-CD8, anti-CD4, anti-IL-2, anti-TNF-α, anti-IFN-γ (BD biosciences), and with viability dye (Life technologies). Flow cytometry was performed using a four-lasers Fortessa (BD biosciences) and analyzed with FlowJo v10 (TreeStar).
6
Multiparametric Characterization of Immune Cells
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The freshly isolated splenocytes, lymphocytes, or BAL cells were stained for 20 min at room temperature using the following fluorochrome-labeled specific antibodies: Alexa Fluor 700 antimouse CD3 (Clone: 17A2; BD), antigen-presenting cell (APC)-labeled antimouse CD8 (Clone: 53-6.7; BD), fluorescein isothiocyanate (FITC)-labeled antimouse CD45.1 (Clone: A20; Biolegend), phycoerythrin (PE)-labeled antimouse CD69 (Clone: H1.2F3; BD), PerCP-Cy5.5-labeled antimouse CD103 (Clone: M290; Biolegend), Brilliant Violet 421-labeled antimouse CXCR3 (Clone: CXCR3-173; Biolegend), FITC-labeled antimouse CD44 (Clone: IM7; BD), and PE-labeled antimouse LPAM-1(α4β7) (Clone: DATK32; BD). A viability dye (Life Technologies) was also included in the staining mix to differentiate living and dead cells. The stained samples were subjected to running on BD LSRFortessaTM instrument followed by analysis with FlowJo X software (Tree Star, Inc.).
7
Resolvin E1 Modulates Inflammation in Mice
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Dorsal air pouches were formed on male Balb/c mice (6 to 8 weeks old) by injecting subcutaneously 3 ml of sterile air in day 0 and day 3. Mice were then intraperitoneally injected with anti-ChemR23 mAb or hIgG1 control mAb at 1 mg/kg or with RvE1 (50 μg/kg) on successive days at d5 and d6. On day 6, inflammation was induced by intrapouch injection of recombinant murine TNF (50 ng) or carrageenan (1%/1 ml per pouch). Pouch lavages (PBS-EDTA 2 mM) were collected at 4, 8, 24, or 48 hours, and cells were stained for phenotyping by flow cytometry. F4/80 (BM8), Ly6G (1A8), and anti-mouse ChemR23 (clone 477806) were used to identify macrophages, PMN, and ChemR23 expression, respectively. PMN mortality was evaluated with a viability dye (Life Technologies). The extraction protocol and analysis of bioactive lipids were performed as previously described (88 ) and adapted according to the Ambiotis SAS (Toulouse, France) standard operating procedure. The liquid chromatography–tandem mass spectrometry experiment was performed on an ExionLC™ AD Ultra-High-Performance Liquid Chromatography system (Sciex) coupled to a QTRAP 6500+ MS (Sciex) and equipped with electrospray ionization source and performed in negative ion mode.
8
Isolation and Phenotyping of Murine Colon Immune Cells
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To collect colonic immune infiltrating cells, colon pieces were incubated in 10 ml of solution 1 [PBS, 30 mM EDTA (ethylenediaminetetraacetic acid), and 1.5 mM dithiothreitol] for 10 min at 37°C under agitation. After filtration, colon pieces were treated with 10 ml of solution 2 (PBS and EDTA 30 mM) for 10 min at 37°C under agitation and lastly filtered and incubated in 5 ml of collagenase D (Sigma-Aldrich) and 25 μl of deoxyribonuclease I (Sigma-Aldrich) solution for 15 min at 37°C under agitation. Splenocytes and bone marrow cells were isolated after lysis of red blood cells, and immune cells were immunophenotyped. Cell mortality was evaluated with a viability dye (Life Technologies). Leukocytes were stained with fluorochrome-conjugated anti-mouse antibodies: F4/80 (BM8), CD11b (M1/70), I/Ab (AF6-120.1), CD3e (500A8), CD4 (RM4-5), CD8 (53-6.7), NK1.1 (PK136), Ly6G (1A8), and CD19 (1D3), all from BD Pharmingen and anti-mouse ChemR23 (clone 477806) from R&D Systems. Fluorescence-activated cell sorting analysis was conducted using a BD Pharmingen LSR II, Canto II flow cytometers, and FlowJo software (Tree Star).
9
Rectal Immune Cell Characterization
10
Rectal Immune Cell Characterization
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Single-cell suspensions from rectal pinches were prepared as previously described30 (link). Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described27 (link). For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.
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