MHC class II tetramers specific for InsB:9–23, GAD:206–220, and human CLIP along with MHC class I tetramers specific to InsB:15-23 and TUM peptide (KYQAVTTTL) were provided by the NIH Tetramer Core Facility (Emory University, Atlanta, GA, USA). Single cell suspensions of cells from lymphoid, pancreatic tissues, or whole blood were prepared as described above. One million cells were added to FACS tubes for preparation of compensation controls. Cells were washed and supernatants were discarded before being resuspended in 200 µL of DPBS (Gibco) plus 10% FBS (Atlanta Biologicals). To block nonspecific staining via Fc receptors, 1.25 µg of anti-mouse CD16/32 (ThermoFisher) was added to each tube. Samples were incubated for 10 minutes at RT, protected from light. Without washing, 0.63 µg of a given tetramer (NIH Tetramer Core Facility) was added to each tube. Samples were incubated at RT for 45 minutes, protected from light. Without washing, cells were stained for surface antigens as described above. Samples were then washed and analyzed on a BD Fortessa flow cytometer without fixation.
Anti mouse cd16 32
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse CD16/32 is a laboratory reagent used for the detection and analysis of mouse cells expressing the CD16 and CD32 antigens. It can be used in various cell-based assays and flow cytometry applications to identify and characterize immune cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Rbc lysis buffer, by Thermo Fisher Scientific (6 mentions)
Facsaria 2 flow cytometer, by BD (5 mentions)
Canto 2, by BD (4 mentions)
Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Cd62l apc cy7, by BioLegend (4 mentions)
Zombie aqua, by BioLegend (3 mentions)
Facsdiva, by BD (3 mentions)
Anti ly6g pe clone 1a8, by BD (3 mentions)
Anti f4 80 fitc clone bm8, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Rorγt pe, by Thermo Fisher Scientific (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Cd3 apc, by BioLegend (3 mentions)
Cd4 pe cy7, by BioLegend (3 mentions)
Foxp3 pe, by BioLegend (3 mentions)
Accuri c6 flow cytometer, by BD (3 mentions)
Cd11c apc, by BioLegend (3 mentions)
Cd8 fitc, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
52 protocols using anti mouse cd16 32
1
Tetramer-based Immune Cell Profiling
2
Characterization of Myeloid Cells by Flow Cytometry
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To carry out flow cytometry analysis, the Fc receptors were initially blocked using anti-mouse CD16/32 (0.25 μg; ThermoFisher) for 15 min at 4°C. Cells were then washed with FACS buffer and stained for surface marker for 30 min at 4°C using the specified antibodies. These antibodies included: CD45 (clone 30-F11), CD11b (clone M1/70) and TGF-β1 (clone TW7-16B4) (all from Biolegend). Cells were then washed with PBS and viability staining was performed using the LIVE/DEAD fixable dead cell stain kit (Invitrogen). Following viability staining, cells were washed with PBS and resuspended in FACS buffer for Flow cytometry analysis. Cells were acquired on a BD Canto II and analyzed using FlowJo X software (vX10). As controls, fluorescence minus one (FMOs) were used to place the gates for analysis. For flow cytometry analysis, cells were first gated according to FSC-SSC, then restricted to singles cells and live cells. Myeloid cells were identified as CD45+ CD11b+.
3
Multicolor Flow Cytometry for Immune Cell Profiling
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Single cells were incubated with anti-mouse CD16/32 (Thermo Fisher) to block Fc receptors and stained as indicated. Lineage cocktail contained (±CD3, ± NK1.1, TCRβ, CD5, CD19, CD11b, CD11c, FcεR1α, F4/80, Ly-6C/G, and Ter119). For intracellular staining we used the Foxp3/Transcription Factor Kit (Thermo Fisher). For intracellular cytokine detection, single cells were stimulated with PMA (60 ng/ml) and ionomycin (500 ng/ml) and 1X protein transport inhibitor (Thermo Fisher) in culture media (RPMI-1640, 10% FCS) at 37°C for 3 hours before staining. Flow cytometry analysis was performed on a BD Fortessa instrument. Cells were quantified using CountBright beads or manual cell counting by hemocytometer. Flow cytometry data was analyzed using FlowJo X (Tree Star).
4
Isolation and Surface Marker Analysis of Mouse Splenocytes
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Splenocytes were isolated from fresh whole mouse spleen. Briefly, the spleen was smashed in PBS with the plunger from a 10‐mL syringe, and the homogenate was passed through a 70‐μm cell strainer. Erythrocytes were lysed with Red Blood Cell Lysis Buffer. Cell suspension was washed with 10–20 mL cold PBS, centrifuged at 400–600 g for 5 min at 4°C, and cell pellet was resuspended in PBS at 1 × 107 cells mL−1. For antibody staining, cells were incubated with antimouse CD16/32 (1 μL mL−1; Thermo Fisher, USA) for 5 min to block non‐specific Fc receptor–mediated antibody binding. 1 × 106 cells were then transferred into polystyrene tubes. Fluorochrome‐conjugated antibodies or isotype controls (0.5 μg/tube) were added, incubated at 4°C for 30 min and washed with PBS. After centrifugation at 300 g for 5 min, cell pellets were fixed with 1% paraformaldehyde, and analysed by flow cytometry on a BD Accuri C6 Plus System. Antibodies used for surface marker labelling included mouse CD3‐FITC, mouse NKp46‐PerCP/Cy5.5, CD8‐FITC from BioLegend (San Diego, USA) and CD44‐PE‐Cy5 from Thermo Fisher. Appropriate isotype‐specific Abs were used as controls. The gating strategy is described in Supplementary figure 6 .
5
Quantifying Dendritic Cells and Th17 Cells
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Single-cell suspensions were initially blocked with an Fc receptor blocker, anti-mouse CD16/32 (Thermo Fisher Scientific, Waltham, MA), on ice for 10 minutes, then stained with fluorochrome-conjugated antibodies for 15 minutes in the dark in accordance with previously established protocols (18 (link), 19 (link)). All flow cytometry analyses were conducted exclusively on the live cell populations, which was achieved by excluding dead cells through staining with Zombie Aqua (BioLegend, San Diego, CA). To quantify type 1 conventional dendritic cells (cDC1) and T helper-17 (Th17) cells, the following anti-mouse primary antibodies were utilized: CD45-APC/Cy7, CD11c-BV421, CD11b-AF700, MHCII-FITC, Ly6C-APC-Cy7, Ly6G-PE, CD3-FITC, CD4-APC, FOXP3-BV421, and RORγT-PE (BioLegend). Activated cDC1 cells were identified as CD45+ Ly6G- MHCII+ CD11c+ CD11b- CD86+. Th17 cells were identified as CD45+ CD3+ CD4+ RORγT+. Analysis was carried out using a BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA), and flow cytometry data were subsequently analyzed using the FlowJo software (FlowJo, Ashland, OR).
6
Multifunctional Nutrient Combination Effects
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Ginseng extract (Ginsenosides ≥80%, designated as R) and EGCG (designated as E) with purity higher that 94% were purchased from Shanghai Novanat Co., Ltd. (Shanghai, China). PDX (designated as D) with purity higher that 90% was purchased from Tate & Lyle Trading Co., Ltd. (Shanghai, China). The complex of selected nutrients was proposed based on the experimental design and designated as the combination of the abbreviation of the corresponding nutrient (ERD). Lipopolysaccharide (LPS) was purchased from Santa Cruz Biotechnology (California, United States). Cell Counting Kit-8 (CCK-8) reagent was purchased from Meilunbio (Dalian, China). Anti-mouse CD16/32, APC-conjugated anti-mouse F4/80, FITC-conjugated anti-mouse CD86, and PE-conjugated anti-mouse CD206 antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, United States) for flow cytometry analysis. Recombinant mouse IFN-γ and IL-4 were provided by Thermo Fisher Scientific. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β analysis were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Neutral red was from sourced from Aladdin (Shanghai, China). An apoptosis kit was purchased from Thermo Fisher Scientific. All reagents used in this study were of analytical grade unless otherwise indicated.
7
Isolation and RNA-seq of Mouse NK Cells
8
Murine Liver Lymphocyte Isolation
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After the mouse liver was removed, a mononuclear cell suspension was prepared by mechanical grinding. Percoll (Cytiva, 17,089,101, Sweden) was used for lymphocyte gradient separation. The cells were washed twice in PBS buffer containing 0.2% bovine serum albumin (BSA) and resuspended in PBS buffer for counting cell number with Trypan blue stain (Gibco, 15250-061, USA). Anti-mouse CD16/32 (eBioscience, 14-0161-86, United States) was used to block the Fc receptor. The following antibodies were used for immunophenotyping analysis of the samples: anti-mouse CD3-FITC (BD, 555,274, USA), anti-mouse NK1.1-APC (BD, 550,627, USA), and anti-mouse NKG2D-PE-Cy7(BD, 562,614, USA), Data were obtained by the flow cytometry (DxFLEX, Beckman Coulter, USA) and analysed using FlowJo 10.0 software.
9
Neutrophil Phenotyping by Flow Cytometry
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Seventy microlitres of blood were taken with heparinised glass capillaries from the tail. After blocking unspecific binding with 10% mouse serum and Fc receptor blocking antibodies (anti-mouse CD16/32, eBioscience, UK), the cell surface was stained with conjugated antibodies for CD11b (Pe-Cy7; eBioscience, UK), and Ly-6G (IA8 clone, APC-Cy7; BioLegend) for 45 min at 4°C. Red blood cells were lysed using FACS Lyse (BD Bioscience) for 10 min after which cells were washed and resuspended in 200 µl PBS. Cells were analysed on the FACS Canto flow cytometer (BD Bioscience) using FACS Diva software.
10
Immune Cell Profiling in Spinal Cord Injury
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