Dissected tumors were freshly cut into small pieces and resuspended in culture medium containing collagenase type 2 (1 mg/ml; Worthington Biochemical) and deoxyribonuclease I (0.1 mg/ml; Boehringer Mannheim), incubated at 37 °C with agitation for 1 h, and then filtered through a 40-mm mesh. Samples were centrifuged, washed twice with PBS, and stained for 30 min at room temperature. Antibodies used are detailed in section for reagents. For quantification of absolute number of cells, a defined number of fluorescent beads (Cell Sorting Set-up Beads for UV Lasers, ThermoFisher) was added to each sample before acquisition and used as a counting reference. Acquisition was performed with a BD LSR II Fortessa (BD Biosciences). Data analysis was conducted using FlowJo version 10.6.1 (Tree Star Inc.). For surface HER3 analysis, NTC and IRE1α-ko cells were treated with osimertinib 200 nM for 20 h and then resuspended with enzyme-free dissociation buffer (Gibco), washed 2× with cold PBS and incubated with EV20 (40 min on ice), washed 3× with cold PBS and stained with AF546-conjugated goat anti-human secondary (ThermoFisher, #A21089). Acquisition done with a BD LSR II Fortessa (BD Biosciences). Data analysis using FlowJo version 10.6.1 (Tree Star Inc.).
Lsrii fortessa
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Switzerland
The BD LSRII Fortessa is a flow cytometry system designed for research applications. It is capable of multi-parameter analysis of cells and particles in suspension. The instrument utilizes a variety of lasers and detectors to measure and analyze various properties of the samples, such as size, granularity, and fluorescence intensity.
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Lab products found in correlation
Facscanto 2, by BD (15 mentions)
Facsdiva software, by BD (13 mentions)
Facsdiva, by BD (12 mentions)
Cytofix cytoperm, by BD (8 mentions)
Ionomycin, by Merck Group (7 mentions)
Phorbol myristate acetate (pma), by Merck Group (7 mentions)
Brefeldin a, by Merck Group (6 mentions)
Cytofix cytoperm kit, by BD (6 mentions)
Facsaria 3, by BD (6 mentions)
True nuclear transcription factor buffer set, by BioLegend (5 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions)
Fc500, by Beckman Coulter (5 mentions)
Facsymphony a5, by BD (4 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (4 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (4 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (4 mentions)
Facscalibur, by BD (4 mentions)
Collagenase d, by Roche (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Perm wash buffer, by BD (3 mentions)
296 protocols using lsrii fortessa
1
Quantification of Surface HER3 Expression
2
Analyzing Virus-Induced Apoptosis and T-Cell Subsets
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To measure the effects of RosA on virus-mediated apoptosis, both floating cells in the culture supernatant and adherent cells detached by EDTA-free trypsin were collected. After washing in 1× annexin binding buffer, cells in 100 µL of binding buffer were incubated with Annexin V-FITC and propidium iodide (PI) at room temperature in the dark for 30 min. Then, cells were analyzed by a flow cytometer (LSRII Fortessa; BD, San Jose, CA) within 4 h.
To analyze the levels of CD3+CD8+ T cells, 20 µL of peripheral blood sample from each group was preincubated with anti-FcγRIII/II (Fc block) (Anti-Mouse CD16/32; 553141). After 30 min of incubation, the following fluorochrome-labelled antibodies: PeCY-7-conjugated CD3 (Clone: 145-2C11; Biolegend), PE-conjugated CD4 (Clone: GK 1.5; Biolegend) and APC-conjugated CD8 (Clone: 53-6.7; Biolegend), were added to the samples for a further 30 min of incubation at room temperature. Afterward, ammonium-chloride-potassium (ACK) lysing buffer was used to lyse red blood cells in the blood samples. Finally, samples were resuspended in 500 µL of staining buffer and analyzed by a flow cytometer (LSRII Fortessa; BD, San Jose, CA).
To analyze the levels of CD3+CD8+ T cells, 20 µL of peripheral blood sample from each group was preincubated with anti-FcγRIII/II (Fc block) (Anti-Mouse CD16/32; 553141). After 30 min of incubation, the following fluorochrome-labelled antibodies: PeCY-7-conjugated CD3 (Clone: 145-2C11; Biolegend), PE-conjugated CD4 (Clone: GK 1.5; Biolegend) and APC-conjugated CD8 (Clone: 53-6.7; Biolegend), were added to the samples for a further 30 min of incubation at room temperature. Afterward, ammonium-chloride-potassium (ACK) lysing buffer was used to lyse red blood cells in the blood samples. Finally, samples were resuspended in 500 µL of staining buffer and analyzed by a flow cytometer (LSRII Fortessa; BD, San Jose, CA).
3
Intracellular Staining for Flow Cytometry
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ICS was performed after live/dead and surface staining, using the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend #426803) according to the manufacturer’s protocol. Briefly, cells were fixed in the Cyto-Fast™ Fix/Perm Buffer for 20 min at room temperature, washed twice in 1X Cyto-Fast™ Perm Wash solution and stained in 1X Cyto-Fast™ Perm Wash solution for 20 min (1:50 dilution) at room temperature. After staining, cells were washed 3× in FACs buffer before acquisition on the BD Fortessa LSRII. For FOXP3 staining, cells were fixed and permeabilized using the True-Nuclear™ Transcription Factor Buffer Set (BioLegend #424401) according to the manufacturer’s protocol, after which FOXP3 was stained for 30 min at room temperature. Cells were then washed and acquired on the BD Fortessa LSRII.
4
Immunophenotyping and Metabolic Assays of PBMCs
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PBMCs were resuspended in 200 μl of FACs buffer. A cocktail of the fluorophores (1:400) were added to stain for the different monocyte subsets. These consisted of Lin (PE—CD2, PE—CD15, PE—CD56, PE—Nkp46, PE−19), APC—HLA-DR, PB—CD14, PE/Cy7—CD16. To measure CD11b levels or GLUT-1 expression, FITC- CD11b (1:400), and FITC—GLUT-1 was also added, respectively. After incubating on ice for 30 min, they were then washed and transferred into FACS tubes. In order to measure metabolism of cells via flow cytometry, 10 nM MitoTracker Deep Red, 4 μM 2-NBDG, 5 μM MitoSOX, and 10 μM H2DCFDA were stained in RPMI 1640 and incubated in 37°C for 20 min before they were washed and transferred into FACS tubes. Cells were immediately run on the BD LSRII Fortessa (BD Biosciences). 100,000 cells were collected for analysis. Unstained and single stained controls were used to set up voltages to compensate for spectral overlap. Flow cytometry data were quantified using FlowJo vX0.7 (FlowJo LCC) software.
5
Intracellular Detection of IL-17 in Th17 Cells
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For intracellular detection of IL-17, CD4+ T cells were incubated with 25 ng/mL phorbol 12-myristate 13-acetate (PMA), 250 ng/mL ionomycin (Sigma-Aldrich), and monensin-containing GolgiStop (BD Biosciences, San Jose, CA, USA) for 4 h. The harvested cells were stained with PerCP-conjugated anti-CD4 antibodies (Biolegend, San Diego, CA, USA). After fixation with fixation/permeabilization solution, the cells were stained with 0.125 μg FITC-conjugated anti-IL-17 antibodies (eBioscience, San Diego, CA, USA) to determine the population of Th17 cells. All analyses were performed using a BD LSRII fortessa (BD Biosciences) and FACS DIVA version 10.0 (BD Biosciences).
6
Isolation and Characterization of Liver Immune Cells
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Single cell preparations from the liver were generated by a combination of mechanical disruption (GentleMACS, Miltenyi Biotec, Bisley, UK) and enzymatic digestion (2 mg/ml Collagenase D, Roche). These were then passed through a 70 μm filter and centrifuged at 50g for 5 min to remove debris and hepatocytes. Red cells were lysed (Red Cell Lysis Buffer, Sigma-Aldrich, Poole, UK). Immune cells were isolated by positive selection using a CD45 + MicroBead AutoMACS separation (Miltenyi Biotec, Bisley, UK) and then stained with a fixable Live-Dead marker (Life Technologies, Paisley, UK) and a multi-colour panel of antibodies, including CD3, CD4, CD8, CD19 and NK1.1 (Biolegend, San Diego, USA). NKT cells were also identified using PBS-57 tetramers, an analogue of alpha-galactosylceramide developed by Dr. Paul Savage (The NIH Tetramer Facility, Emory University, Atlanta, USA) and complexed to CD1d tetramers [42] (link).
Samples were then run on a BD LSR II Fortessa (BD Biosciences, Oxford, UK) and analysed with FlowJo software (Tree Star, Ashland, USA). T cells were defined as CD3 + CD19 − cells and NKT cells as CD3intNK1.1 + (or CD3 + Tetramer +) cells, within a forward-side scatter defined lymphocyte gate (Supplemental Fig. 1).
Samples were then run on a BD LSR II Fortessa (BD Biosciences, Oxford, UK) and analysed with FlowJo software (Tree Star, Ashland, USA). T cells were defined as CD3 + CD19 − cells and NKT cells as CD3intNK1.1 + (or CD3 + Tetramer +) cells, within a forward-side scatter defined lymphocyte gate (Supplemental Fig. 1).
7
Quantifying Cytokine Responses in Splenocytes
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Monoclonal antibodies Brilliant Violet 421 anti-mouse CD3 clone 17A2, PE anti-mouse CD4 clone GK1.5, Brilliant Violet 650 anti-mouse CD8 clone 53-6.7, APC anti-mouse IFN-γ clone XMG1.2, PE/Cy7 anti-mouse TNF-α clone MP6-XT22, and Alexa Fluor 488 anti-mouse interleukin-2 (IL-2) clone JES6-5H4 were obtained from BioLegend (San Diego, CA, USA). Intracellular cytokine staining was performed as described previously [42 (link)]. Briefly, 2 × 106 splenocytes were plated into each well and stimulated with 5 μg/mL HA peptide pool or DMSO (negative control) for 16 h. Then, 5 h prior to the end of stimulation, 1 μg/mL brefeldin A (BioLegend) was added to each well. Cells were then washed with FACS buffer (2% FBS in sterile DPBS) and surface staining was performed in the dark on ice for 30 min. Cells were fixed with IC FIX buffer (BioLegend), and permeabilized with 1X permeabilization buffer (BioLegend). Intracellular cytokine staining was then performed in the dark on ice for 40 min. The stained samples were then washed twice with FACS buffer and fixed with 0.5% PFA. For each sample, 1 × 106 events were acquired on a BD LSRII Fortessa, and data were analyzed using Flowjo software V10.
8
Monocyte NF-κB Signaling Dynamics
9
Flow Cytometry Analysis of Transfected Cells
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HEK293T and MEF-Ai6 cells were trypsinized and collected in PBS with 10% fetal bovine serum 24 and 72 h after transfection, respectively. Cells were analyzed on a BD LSRII Fortessa. ZsGreen fluorescent protein was excited using a 488-nm laser and detected using a 525/50 filter. mCherry fluorescent protein was excited using a 561-nm laser and detected using a 610/20 filter. Single color controls are shown in Supplementary Figure S8 . For HEK293T, a minimum of 20 000 and for MEF-Ai6, a minimum of 5000 mCherry-positive events were recorded. Data analysis was performed using FlowJo software, and flow plots were processed using Adobe Illustrator.
10
IL-33 Stimulates ILC2 Cell Analysis
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1 × 104 ILC2 cells were stimulated with IL-33 (100 ng/ml) for 48 hours. Following stimulation cells were stained with Fixable Viability Dye eFluor™ 450 (eBioscience, California, United States) for 10 minutes at 4 °C according to manufacturer instructions. Cells were fixed in ice cold 70% ethanol, washed twice with PBS. Samples were then incubated for 5 min with RNase A (100 μg/ml) (Thermo Fisher Scientific), stained with Propidium iodide (50 μg/ml) (Sigma-Aldrich) and analysed using BD LSRII Fortessa (BD). Results were further analysed by FlowJo software (Tree Star).
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