Spectral flow fcs files were initially analysed using SpectroFlo v3.0 (Cytek) for unmixing and autofluorescence correction. Identification of T cell subsets from unmixed fcs files was performed using Flowjo v10 (Flowjo LLC). Antigen specific cytokine levels are reported after subtraction of values of unstimulated cells for each participant. K/T ratio was determined from the concentrations of tryptophan and kynurenines after quality control checks. Non-parametric tests were used for comparison between two groups for paired (Wilcoxon test) or unpaired (Mann-Whitney test) and p < 0.05 considered significant. Adjusted comparisons between groups in continuous variables were performed with linear regression, transforming outcome variables to satisfy model assumptions. Spearman correlation was used to test the association between parameters with a p < 0.05 considered significant. All statistical analyses used Prism 9.0 (GraphPad).
Spectroflo v3
Manufactured by Cytek
Sourced in United States
The SpectroFlo v3.0 is a multiparameter flow cytometer developed by Cytek. It is designed to analyze and sort cells based on their physical and fluorescent characteristics. The instrument is capable of detecting and measuring multiple parameters simultaneously, providing detailed information about the properties of individual cells within a sample.
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Lab products found in correlation
5 protocols using spectroflo v3
1
Multiparametric Analysis of Immune Responses
2
Characterization of T-cell Subsets
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Spectral flow fcs files were initially analyzed using SpectroFlo v3.0 (Cytek Biosciences, Fremont, California, USA) for unmixing and autofluorescence correction. Identification of T-cell subsets from unmixed fcs files was performed using Flowjo v10 (Flowjo LLC, Ashland, Oregon, USA). Antigen-specific cytokine levels are reported after subtraction of values of unstimulated cells for each participant. K/T ratio was determined from the concentrations of tryptophan and kynurenines after quality control checks. Non-parametric tests were used for comparison between two groups for paired (Wilcoxon test) or unpaired (Mann–Whitney test) and p< 0.05 was considered significant. Spearman correlation was used to test the association between parameters with a p< 0.05 considered significant. All statistical analyses used Prism 9.0 (GraphPad, Boston, Massachusetts, USA).
3
Hepatic Leukocyte Subset Analysis
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To analyze hepatic leukocyte subsets, isolated CD45+ leukocytes were incubated with a cocktail of antibodies directed against CD11c, CD19, Ly6G, F4/80, MHC-II, CLEC2, Siglec-F, CD64, NK1.1, CD11b, Ly6C, CD3, Thy1.2 and TIM4 (details regarding the antibodies are presented in Supplementary Table S2 ) in PBS/BSA/EDTA supplemented with True-Stain monocyte blocker (426103, Biolegend) and Brilliant Stain Buffer Plus (566385, BD biosciences) for 30 min at 4 °C. The stained samples were measured by spectral flow cytometry using a 3-laser Cytek Aurora spectral flow cytometer (Cytek Biosciences). Spectral unmixing was performed using SpectroFlo v3.0 (Cytek Biosciences). Gating of flow cytometry data was performed using FlowJoTM v10.8 Software (BD Biosciences). A representative gating strategy is presented in Supplementary Fig. S2A .
4
Spectral Flow Cytometry Analysis of Immune Cell Populations
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In experiment 2, after 15 weeks of treatment, 8 mice per group were selected, based on having body weight, lean mass and fat mass close to the group mean, and 12-hour-fasted blood was collected from the retro-orbital plexus just after the mice were killed by CO2 inhalation. In experiment 3, after 12 weeks of treatment, 4-hour-fasted blood was collected via a tail vein cut. Obtained blood samples were lysed for 20 min at room temperature using an erythrocyte lysis/fixation solution (BD Biosciences, USA). Leukocytes were then centrifuged at 596 rcf for 5 minutes at 4°C, reconstituted in PBS, and washed three times in PBS. Isolated leukocytes were treated with a cocktail of antibodies (details are provided in Tables S2 and S3 ) in PBS containing 2 mM EDTA, 0.5% BSA, True-Stain monocyte blocker (Biolegend, USA) and Brilliant Stain Buffer Plus (BD Biosciences, USA) for 30 minutes at 4°C, as previously reported.52 (link) After staining, samples were analyzed by spectral flow cytometry using a Cytek Aurora Spectral flow cytometer (Cytek Biosciences, The Netherlands) or a Cytoflex S (Beckman Coulter, Woerden, the Netherlands). SpectroFlo v3.0 (Cytek Biosciences, The Netherlands) and FlowJoTM v10.8 software (BD Biosciences, USA) were used for spectral unmixing of the flow cytometry data and gating of the data, respectively. A representative gating strategy is shown in Figures S3 and S4 .
5
Spectral Flow Cytometry Analysis of Murine Leukocytes
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Whole blood samples were diluted 1:1 in PBS and treated with erythrocyte lysis/fixation solution (349202; BD Biosciences) for 15 min at room temperature. After lysis, cells were pelleted by centrifugation (5 min; 596 g; 4 °C) and washed 3 times with PBS. Isolated leukocytes were subsequently incubated with a cocktail of antibodies directed against CD3, CD11b, CD11c, CD19, CD36, CD45, CD62L, Ly6C, Ly6G, NK1.1, Siglec-F and TREML4 (details regarding the antibodies are presented in Table S4) in PBS supplemented with 0.5% BSA, 2 mM EDTA, True-Stain monocyte blocker (426103; Biolegend) and Brilliant Stain Buffer Plus (566385; BD Biosciences) for 30 min at 4 °C. Stained samples were analyzed by spectral flow cytometry using a Cytek Aurora Spectral flow cytometer (Cytek Biosciences). SpectroFlo v3.0 (Cytek Biosciences) was used for spectral unmixing of the flow cytometry data, and FlowJo™ v10.8 software (BD Biosciences) was used for gating of the data. A representative gating strategy is depicted in Supplementary Fig. S1A.
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