PBMCs were obtained and labeled with CFSE as previously described [43 (link)]. The labeled PBMCs were resuspended in RPMI-1640 medium supplemented with 10% FBS at final concentration of 5 × 106 cells/mL. All cells were incubated in 48-well flat-bottomed plates at a density of 3 × 106 cells/well in 0.5 mL of culture medium, and then stimulated with SEC2/ST-4 at a final concentration of 100 ng/mL for 72 h. Untreated PBMCs served as negative control. After incubation, cell division analysis was performed using BD Biosciences LSRFortessa, and data were analyzed with FlowJo V10 software (Tree Star, Ashland, OR, USA). % Divided is the percentage of the cells of the original sample that divided.
Lsr fortessa
Manufactured by Tree Star
Sourced in United States
The LSR Fortessa is a flow cytometry instrument designed for advanced multiparameter analysis. It features a compact design and incorporates up to 5 lasers and 18 detectors to enable the simultaneous detection of multiple cell parameters. The core function of the LSR Fortessa is to provide high-performance data acquisition and analysis capabilities for a wide range of applications in flow cytometry.
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Lab products found in correlation
Fixable viability dye, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
Perm wash buffer, by BD (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Facs canto, by Tree Star (2 mentions)
Live dead aqua 525, by Thermo Fisher Scientific (2 mentions)
Dq ovalbumin, by Thermo Fisher Scientific (2 mentions)
Canto 2, by BD (1 mentions)
Cd16 cd32 rat anti mouse antibody clone 2.4g2, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Nylon cell strainer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Liberase ci, by Roche (1 mentions)
Anti mouse cd16 cd32, by BD (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Fluorochrome conjugated mabs specific, by BioLegend (1 mentions)
Cd11b bv650 clone m1 70, by BioLegend (1 mentions)
Cd11c apc, by BD (1 mentions)
25 protocols using lsr fortessa
1
CFSE-Labeled PBMC Proliferation Assay
2
Multicolor Flow Cytometry Staining
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Single-cell suspensions from tissues and cells from BAL fluid were stained for surface markers for 30 min at 4°C in PBS containing 0.01% (m/v) sodium azide and 1% (m/v) BSA. Propidium iodide (0.3 μg/mL, Sigma, St. Louis, MO, USA) in PBS was used to distinguish vital cells. Samples were treated with rat anti-mouse CD16/CD32 antibody (clone 2.4G2, BD Biosciences, Basel, Switzerland) to block FcR-mediated non-specific antibody binding prior to incubation with fluorochrome-conjugated antibodies. Acquisitions were made with a BD Canto II or LSR Fortessa and analyzed using FlowJo software (version 10.0.7, Treestar).
3
Flow Cytometric Analysis of DC Maturation
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To analyze the expression of DC maturation and T-cell markers, the DCs and T cells were trypsinized and fixed with 4% PFA. Staining for cell-surface markers was performed by incubating cells with conjugated primary antibodies (Supplementary Table S3) diluted at a 1:200 ratio in FACS buffer (0.1% BSA in PBS) for 30 minutes at 4°C. Samples were then processed using LSRFortessa and analyzed with FlowJo software (v10.8.1; Tree Star).
4
Comprehensive Immune Phenotyping of T Cells
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Lymphocytes were transferred into FACS tubes and stained with fluorescence-conjugated monoclonal antibodies as follows; anti-human BV785-conjugated anti-CD3 (1:100; Biolegend, # 344842), APC-Fir750-conjugated anti-CD4 (1:100; BD, # 563800), BV510-conjugated anti-CD8 (1:100; Biolegend, #344732), PE-CF594-conjugated anti-CD25 (1:100; Biolegend, #356126), PE-conjugated anti-CD127 (1:100; Biosciences, #557938), AF700-conjugated anti-CD45RA (1:100; Biolegend, #304120), BV421-conjugated anti-CCR7 (1:25; Biolegend, # 353208), FITC-conjugated anti-CD38 (1:100; eBioscience, #11-0388-42), BV605-conjugated anti-HLA-DR (1:100; Biosciences, #564017), BV711-conjugated anti-PD-1 (1:100; Biosciences, #564017), PE-CY7-conjugated anti-TIGIT (1:100; eBioscience, #25-9500-42), BV650-conjugated anti-Tim-3 (1:100; Biosciences, #565564), APC-conjugated anti-CTLA4 (1:100; Biosciences, #349908), and corresponding isotype controls. Data were acquired using a LSR Fortessa flow cytometer and analyzed with FlowJo software (Tree Star).
5
Isolation and Flow Cytometry Analysis of Muscle-Resident Macrophages
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Single-cell suspensions of hind-limb muscles containing the femoral arteries, and paw tissues were generated by careful mincing tissues and subsequent digestion at 37°C for 1 hour in DMEM (Life Technologies) in the presence of 250 µg/ml liberase CI (Roche) plus 50 µg/ml DNase I (Roche). After filtration through a 40 µm nylon cell strainer (BD Biosciences) and centrifugation for 5 min at 400 g, the cells were washed and treated with ammonium-chloride-based erythrocyte lysis buffer. All flow cytometric measurements were performed in the buffer containing PBS with 2 mM EDTA and 2 % FBS (Life Technologies) on a Becton Dickinson LSRFortessa flow cytometer and were analyzed by FlowJo software (Treestar Inc.). Csf1r-EGFP positive and MF800 positive signals have been detected through FITC/Alexa Fluor488 (Excitation: 488 nm laser with 50 mW power; Emission: 530 ± 15 nm filter) and APC-Cy7/APC-H7 (Excitation: 640 nm laser with 40 mW power; Emission: 780 ± 30 nm filter) channels, respectively.
6
Staining for Splenic Myeloid Cells
7
Flow Cytometric Immune Cell Analysis
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Single-cell suspensions from spleens or draining inguinal lymph nodes (dLNs) were incubated with biotinylated antibodies (S1 Table ) for 20 minutes on ice, washed twice with 200 μl PBS supplemented with 2% FBS, 1 mM EDTA, and 0.1% NaN3 (FACS buffer), incubated with fluorophore-conjugated antibodies and streptavidin (S1 Table ) for 20 minutes on ice, washed twice more with 200 μl FACS buffer, and resuspended in FACS buffer for acquisition. For intracellular staining, surface-stained cells were fixed and permeabilized for 20 minutes on ice with BD Cytofix/Cytoperm buffer, washed twice with 200 μl BD Perm/Wash buffer, incubated with for 20 minutes on ice with fluorophore-conjugated antibodies (S1 Table ), followed by two washes with 200 μl Perm/Wash buffer, and resuspended in FACS buffer for acquisition. Data were acquired on a FACSCanto or LSRFortessa and analyzed using FlowJo (TreeStar).
8
Influenza NP-specific CD8+ T cell analysis
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MHC‐I tetramer targeting the immunodominant epitope of the influenza nucleoprotein (DbNP366–374 – ASNENMETM, DbPA224–233 – SSLENFRAYV) was produced in‐house and conjugated to streptavidin‐APC/PE (Life Technologies Australia Pty Ltd) at a 1:250 dilution at room temperature for 1h. Cells were stained with combinations of fluorochrome‐conjugated antibodies: PerCP‐Cy5.5‐CD3 (#551163), PE‐CD8 (#561095), BV421‐I‐Ab (#562928), APC‐CD44 (#553133), FITC‐CD44 (#553133), BV711‐CD38 (#740697), PerCP‐Cy5.5‐CD8 (#551162), APC/eF780‐CD62L (#47‐0621‐82), APC‐CD11c (#17011481), PE‐Cy7‐TCR‐va2 (#560624) from BD Biosciences, USA; and PE‐Cy7‐CD38 (#102718), BV785‐PD‐1 (#329908), APC‐Cy7‐CD45.1 (#110716), FITC‐I‐Ab (#116406), Pacific Blue‐I‐Ab (#116422), FITC‐CD19 (#115506) from Biolegend, USA. AF700‐CD3 (#56003382) was purchased from Invitrogen, USA.
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
9
Microglia Isolation and Flow Cytometry
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To isolate microglia, cells were first Fc-blocked with anti-CD16/32 (1:200; BD Biosciences). Cells were then stained with anti-CD11b APC (1:200; BioLegend), anti-CD45 BV786 (1:200; BD Biosciences), anti-CD11c PE (1:400; BioLegend), and anti-MHCII BUV737 (1:200; BD Biosciences) for 30 min at 4°C. Cells were washed with PBS and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (1:500; Invitrogen) for 20 min. CD11b+/CD45inter microglia were sorted using a FACSAria Fusion (BD Bioscience). Cells were washed and stained for LIVE/DEAD Fixable far-red dead Cell Stain (1:500; Invitrogen) for 20 min and washed with PBS. Cells were analyzed on LSR Fortessa and FlowJo software (Tree Star).
10
Single-cell flow cytometry protocol
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