Peripheral blood was obtained from retro-orbital puncture, and red blood cells were lysed by ACK lysis buffer. CD45 (BioLegend, 109820, 1:200) staining was performed at 4 °C for 30 min. Flow cytometry analysis was performed on Fortessa machines (BD Bioscience), and data were analyzed with FlowJo.
Fortessa machine
Manufactured by BD
Sourced in United Kingdom, United States
The BD Fortessa is a multi-parameter flow cytometer designed for analyzing and sorting cells. It is capable of detecting and measuring various characteristics of individual cells, including size, granularity, and the presence of specific proteins or molecules on the cell surface or within the cell. The BD Fortessa is a versatile instrument that can be used for a wide range of applications in fields such as immunology, cell biology, and stem cell research.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Phospho s6 s235 236, by Cell Signaling Technology (1 mentions)
Cd25 pc61, by BD (1 mentions)
Cd127 a7r34, by Thermo Fisher Scientific (1 mentions)
Cd44 im7, by BioLegend (1 mentions)
Bcl 6 k112 91, by BD (1 mentions)
Cd62l mel 14, by BD (1 mentions)
Cd4 rm4 5, by BD (1 mentions)
T bet 4b10, by BioLegend (1 mentions)
Tcf1 c63d9, by Cell Signaling Technology (1 mentions)
Cxcr5 2g8, by BD (1 mentions)
Slam tc15 12f12.2, by BioLegend (1 mentions)
Ccr7 4b12, by Thermo Fisher Scientific (1 mentions)
Zombie aqua dye, by BioLegend (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Facsdiva, by BD (1 mentions)
Anti ifn γ pe, by BioLegend (1 mentions)
Anti cd4 pe cy7, by BioLegend (1 mentions)
Anti tnf α apc, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
11 protocols using fortessa machine
1
Immunophenotyping of Blood Cells
2
Comprehensive Immune Cell Phenotyping
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Single-cell suspensions were prepared, and cells were stained according to standard staining protocol. Surface staining was done with the following antibodies: CD4 (RM4-5; BD), CD127 (A7R34; eBioscience), CCR7 (4B12; eBioscience), CD44 (IM7; BioLegend), SLAM (TC15-12F12.2; BioLegend), CD62L (MEL-14; BD), CXCR5 (2G8; BD), and CD25 (PC61; BD). Intracellular staining was done with the following antibodies: T-bet (4B10; BioLegend), TCF1 (C63D9; Cell Signaling Technology), Bcl6 (K112-91; BD), and phospho-S6 (S235/236; Cell Signaling Technology). Flow cytometry was acquired on LSRII and Fortessa machines (BD), and analysis was performed with FlowJo software (Tree Star).
3
Phenotypic Characterization of EBOV-specific T Cells
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PBMCs were resuspended in warmed complete media and rested overnight at 37 °C & 5% CO2. The following day cells were adjusted to 2×106 cells/ml in media containing anti-CD28, CD49d and CD107a-PerCP cy5.5 (1 µg/ml). Samples were then left either untreated (NT) or were stimulated with EBOV GP peptide pool, containing 187 15 mer overlapping peptides at 2.5 µg/peptide or 1 µg/ml SEB for 16–18 h2 (link),34 (link). Two hours into the incubation brefeldin A and monensin (1 µg/ml) were added to block cytokine secretion from the cell. The following day samples were washed in cold FACS wash and LIVE/DEAD fixable aqua dye was added. Samples were washed, then incubated with a cell surface cocktail of antibodies including CD3-APC 750, CD4-BV786, CD8-AF700, CD19-BV510, CD14-BV510, CCR7- APC, CD95-BV395, CD45RO-BV605. Cells were then washed, fixed and permeabilised before staining for intracellular cytokines using IFNγ-AF488, TNFα- BV421 and IL-2-PE. Samples were then washed resuspended and acquired using a BD Fortessa machine and FACS Diva software (BD Biosciences, UK). Sample analysis utilised FlowJo™ v10 software2 (link),20 (link).
4
Comprehensive Flow Cytometry Staining Protocol
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Unspecific binding sites were blocked using a CD16/CD32 antibody. Dead cells were stained with Zombie Aqua™ dye (BioLegend). The antibodies used for flow cytometry analysis are listed below. Stained cells were quantified using a BD Fortessa machine and BD FACSDivaTM 6.0 software (both from BD Biosciences). FlowJo v10 software (FlowJo) was used for data analysis. Compensation was performed with single‐colour controls. Compensation matrices were calculated using FlowJo v10 software and applied. Doublets and dead cells were excluded from the analysis. Fluorescence minus one (FMO) controls were used for gating analyses to distinguish positively from negatively stained cell populations.
The following dyes and antibodies were used for flow cytometry analysis:
The following dyes and antibodies were used for flow cytometry analysis:
| Antigen | Clone | Fluorophore | Dilution | Source |
|---|---|---|---|---|
| CD45 | 30‐F11 | PB | 1:400 | BioLegend |
| Fc block (CD 16/32) | 2.4G2 | NA | 1:100 | BD BioSciences |
| CD3 | 17A2 | BV785 | 1:300 | BioLegend |
| I‐A/I‐E | M5/114.15.2 | PE | 1:2000 | BD Biosciences |
| TCRδ | GL3 | FITC | 1:400 | BD Biosciences |
5
Murine Splenic Immune Cell Profiling
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Spleens were collected and mononuclear cells isolated via gentle extrusion of the tissue through a 50-μm-mesh nylon cell strainer (BD). Cells were resuspended in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, 50 U/mg penicillin, and 50 U/mg streptomycin. Erythrocytes were lysed with red-blood-cell lysing buffer (Sigma-Aldrich, St. Louis, MO, United States). Cells collected at D28 were first stained with eFluor 506-labeled fixable viability dye (eBioscience), and a surface staining was then performed with APC-eFluor780-labeled anti-CD3 (145-2C11), PECy7-labeled anti-NKp46 (29A1.4), PE-labeled anti-CD4 (RM4–5), FITC-labeled anti-Foxp3 (FJK-16s) and Brillant Violet 605-labeled anti-CD8α (53–6.7) antibodies. Flow cytometry analyses were carried out using a BD-Fortessa machine. Data were analyzed using FlowJo software (version X10.0.7).
6
Cell Cycle Profiling via Flow Cytometry
7
Intracellular Cytokine Staining of PBMCs
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Intracellular cytokine staining was performed as previously described [13 (link)]. Briefly, PBMCs were resuspended in warmed media and rested overnight at 37°C. The following day, cells were adjusted to 1 × 106 cells/mL in media containing anti-CD28, CD49d, and CD107a-PerCP cy5.5 (1 µg/mL). Cells were then untreated or stimulated with EBOV GP peptide pool, containing 187 × 15-mer overlapping peptides at 2.5 µg/peptide or 1 µg/mL Staphylococcal Enterotoxin B peptide for 16–18 hours. After 2 hours, brefeldin A and monensin (1 µg/mL) were added to block cytokine secretion. The following day, samples were washed and LIVE/DEAD dye added. Samples were washed, incubated with cell surface antibodies (CD3-APC 750, CD4-BV786, CD8-AF700, CD19-BV510, and then CD14-BV510, CCR7-APC, CD95-BV395, and CD45RO-BV605); then washed, fixed, and permeabilized; then stained for intracellular cytokines using IFNγ-AF488, TNFα-BV421, and IL-2-PE. Samples were analyzed using a BD Fortessa machine and FACS Diva, FlowJo, Pestle, and SPICE software (see Supplementary Information ).
8
Multiparametric Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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Tumors were dissociated into single cells and were then filtrated through 70 μm cell strainers. Single cells were resuspended in cell staining buffer (#FXP005, 4abio) and then stained with fluorochrome-conjugated antibody combinations at appropriate concentration. The antibody information are shown as follows: PerCP-Cy5.5-Anti-CD45 (#103131, Biolegend), PE-Cy7-Anti-CD4 (#100421, Biolegend), FITC-Anti-CD8 (#ab237367, Abcam), APC-Anti-TNF-α (#506307, Biolegend), PE-Anti-IFN-γ (#505807, Biolegend), PerCP-Cy5.5-Anti-CD45R (#ab210342, Abcam), and PE-Anti-CD3 (#ab22268, Abcam). DAPI (#D9542, Sigma) was added to exclude dead cells. After washing, the stained cells were analyzed on a BD Fortessa machine. The data were processed with FlowJo software.
9
Spleen Dissociation and Cell Staining for Flow Cytometry
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Mouse spleens were minced with scissors in 0.5 mg/mL collagenase complete media, and then incubated at 37° on a nutating mixer for 45 min. The spleen tissues were then homogenized through a 70 um cell strainer and treated with ammonium chloride potassium (ACK) lysis buffer to remove red blood cells. The splenocytes were then washed and counted in trypan blue and resuspended in fluorescence-activated cell sorting (FACS) buffer (0.5% fetal bovine serum in PBS). Cells were plated at 1 × 106 cells/well, and AmCyan live/dead stain (Invitrogen) was used to identify live cells. Cells were then stained for surface markers for 15 min on ice, fixed and permeabilized (Foxp3 fixation/permeabilization kit, Ebioscience), and then stained intracellularly with antibodies for 30 min on ice. Flow cytometry was performed on a BD Fortessa machine using BD FACSDiva software. Analysis was performed using FlowJo software.
Directly conjugated antibodies were used in three separate staining panels. Antibodies were tested using cells from the 8 CC founder strains to confirm that antibody clones were compatible with the CC mice prior to being used for testing (with the exception of MHC Class II, which works only on strains with the correct haplotype).Tables 2 , 3 , and 4 contain antibody names, fluorochromes, and clones for each of the antibodies used.
Directly conjugated antibodies were used in three separate staining panels. Antibodies were tested using cells from the 8 CC founder strains to confirm that antibody clones were compatible with the CC mice prior to being used for testing (with the exception of MHC Class II, which works only on strains with the correct haplotype).
10
Multiparametric Flow Cytometry of MSCs
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Per condition, 2 x 105 MSCs were re-suspended in 500 μl FACSFlow solution (BD Biosciences) and stained with antibodies against human CD45-APC (368515, BioLegend), CD90-APC (FAB2067A, R&D Systems), CD73-PE (550257, BD Biosciences), or CD105-FITC (FAB10971F, R&D Systems), following the manufacturer's guidelines. Afterwards, the cells were fixed using 2% formaldehyde (Fluka) and were filtered through 70-μM filters. Unstained cells were used as a negative control. Samples were analyzed by flow cytometry using a BD Fortessa machine (BD Biosciences). The data were analyzed using FlowJo V10 software.
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