CD4+ T cells were enriched from PBMCs by negative selection using magnetic beads (Miltenyi) and naïve CD45RA+CCR7+CD4+ T cells were then isolated by cell sorting using a BD FACSAria™ cytometer. Naïve CD4+ T cells were seeded at a concentration of 5 × 105 cells per well in a 96 well plate in complete medium and stimulated with anti-CD2/CD3/CD28 beads (Miltenyi) alone (TH0 condition) or in the presence of human recombinant cytokines: For TH1 conditions IL-12 (20ng/mL) (Biolegend); for TH17 conditions IL-6 (20ng/mL), IL-23 (10ng/mL) (Biolegend) and TGF-β1 (5 ng/mL) (R&D Systems); for TFH conditions IL-12 (2ng/mL), IL-23 (10 ng/mL) and TGF-β1 (5ng/mL)35 (link).
Facsaria cytometer
Manufactured by BD
Sourced in United States, United Kingdom, Spain
The FACSAria cytometer is a high-performance flow cytometry instrument designed for cell sorting and analysis. It provides precise data acquisition and single-cell sorting capabilities for a wide range of applications.
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Lab products found in correlation
Facsdiva software, by BD (7 mentions)
Fcap array software, by BD (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Sytox green, by Thermo Fisher Scientific (4 mentions)
Lsr 2, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (3 mentions)
Macs negative selection kit, by STEMCELL (3 mentions)
Anti cd16 32, by Thermo Fisher Scientific (2 mentions)
Anti ccr9, by Thermo Fisher Scientific (2 mentions)
Duoset elisa kit, by R&D Systems (2 mentions)
Luciferin luciferase bioluminescence assay, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc apoptosis analysis kit, by Sungene Biotech (2 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
293ft cell, by Thermo Fisher Scientific (2 mentions)
Profection mammalian transfection system, by Promega (2 mentions)
130 protocols using facsaria cytometer
1
Naïve CD4+ T cell differentiation
2
PKH26 Staining and Flow Cytometry
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PKH staining was performed as previously described (19).
CTSCs were trypsinised, filtered through a 40-μm cell strainer, resuspended in PBS (approximately 500,000 cells/ml), labelled with PKH26 (Sigma, 10−7 M, 5 min), washed twice, and plated.
For the flow cytometric analysis of PKH26-stained cells, CTSCs were trypsinised, filtered through a 40-μm cell strainer, and resuspended in PBS at a concentration of 1×106/ml. The cells were subdivided into 5-ml polystyrene tubes (Falcon, Becton Dickinson). The BD FACSAria cytometer, equipped with four excitation laser lines (633 nm, 488 nm, 405 nm, and 375 nm) (Becton Dickinson) was used for FACS analysis, and the BD FacsDIVA software was used for data analysis.
PKH26 staining was evaluated by selecting the appropriate cell population according to the following gating strategy: (i) cells were first gated on physical parameters (forward scatter [FSC] and side scatter [SSC]) to exclude most of the debris and dead cells; (ii) doublets and aggregates were eliminated using the FSC-area vs. FSC-height pattern. We gated 10-15% of the brightest PKH26+ cells in a PKH26 versus empty channel dot plot.
CTSCs were trypsinised, filtered through a 40-μm cell strainer, resuspended in PBS (approximately 500,000 cells/ml), labelled with PKH26 (Sigma, 10−7 M, 5 min), washed twice, and plated.
For the flow cytometric analysis of PKH26-stained cells, CTSCs were trypsinised, filtered through a 40-μm cell strainer, and resuspended in PBS at a concentration of 1×106/ml. The cells were subdivided into 5-ml polystyrene tubes (Falcon, Becton Dickinson). The BD FACSAria cytometer, equipped with four excitation laser lines (633 nm, 488 nm, 405 nm, and 375 nm) (Becton Dickinson) was used for FACS analysis, and the BD FacsDIVA software was used for data analysis.
PKH26 staining was evaluated by selecting the appropriate cell population according to the following gating strategy: (i) cells were first gated on physical parameters (forward scatter [FSC] and side scatter [SSC]) to exclude most of the debris and dead cells; (ii) doublets and aggregates were eliminated using the FSC-area vs. FSC-height pattern. We gated 10-15% of the brightest PKH26+ cells in a PKH26 versus empty channel dot plot.
3
Cytokine and Flow Cytometry Analysis of T. muris Infection
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Single cell suspensions were prepared from mesenteric lymph nodes (MLNs) taken at autopsy and added at 5 × 106 cells per well in 1ml cultures to 48-well plates and stimulated with T. muris E/S at 50μg/ml. Cells were incubated at 37°C, 5% CO2, 95% humidity for 48 hours, after which time supernatants were harvested and stored at −20°C. For cytokine analysis levels of IL-4, IL-10, IL-6, IL-9, IL-13, Interferon gamma, tumour necrosis factor α and IL-12p70 were determined using a custom cytometric bead array according to manufacturers instructions (CBA, Becton Dickenson, Oxford, UK) and analysed using BD FacsAria cytometer and FCAP Array software. For flow cytometry analysis single cell suspensions were prepared. Total cell numbers were counted and the cells were resuspended at 5 × 106 cells/ml. Cells were washed and Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed anti-CD4, anti-CD8, CD44, CD62L and anti-CCR9 (E bioscience) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).
4
Quantifying Intracellular Oxidative Stress
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Intracellular ROS production was measured using the nonfluorescent DCFH-DA reagent that could be converted to the fluorescent DCF by free radicals [37 (link)–39 (link)]. In brief, the cells were homogenized in the assay buffer and then incubated with DCFH-DA (10 μmol/L) at 37°C for 30 min. The fluorescent intensity was examined using a spectrofluorometer with an excitation/emission wavelength at 488/525 nm. To detect lipid ROS level, the cells were incubated with BODIPY (10 μmol/L) at 37°C for 30 min and the fluorescent intensity was recorded by the simultaneous acquisition of green signals (484/510 nm) and red signals (581/610 nm) using the BD FACSAria cytometer [32 (link)]. Intracellular MDA content was assessed using the commercial kit following the manufacturer's instructions, and the absorbance was measured at 532 nm [32 (link), 40 (link)].
5
iPSC Characterization via SSEA-4
6
CD4 T Cell Enrichment and Sorting
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Splenic cell suspension was prepared as mentioned above and CD4 T cells were enriched by negative selection using magnetic EasySep selection kit (Stemcell Technologies) and biotinylated antibodies against B220, CD19, CD11b, NK1.1, Gr1, Ly6G, and Ter19. CD11ahiCD49dhi or CD11aloCD49dlo CD4 T cell populations were subsequently sorted using a BD FACS Aria cytometer (purity ≥90%). Mice received 1 × 106 cells via intravenous injection.
7
Cytokine Profiling in Mouse Models
8
Flow Cytometry of Apoptosis Induction
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Flow cytometry was performed using standard immunology protocols. Briefly, experimental and control cells were electroporated with respective plasmids. After a defined time point, cells were collected, fixed and permeabilized using Fixation/Permeablization Solution kit (BD). Then antigen-specific antibodies with specific dilutions were added into cells and incubated for 30 min on ice. Cells were washed with cold MACS buffer for 3 times before analyzed on a BD FACSAria cytometer. Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200).
9
Quantifying Intracellular Lipid ROS
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Lipid ROS level was analyzed by flow cytometry using BODIPY-C11 (GLPBIO, GC40165) dye. Cells were seeded at 2.5 × 105 per well in a six-well dish and grown overnight for 12 h. Cells were washed once with PBS. Cells were then stained with 2 ml medium containing 5 µM of BODIPY-C11 and incubated at 37 °C for 20 min in the dark. Cells were washed twice with PBS to remove excess labeling mixture, followed by resuspending in 500 μl fresh PBS (DPBS, Gibco). The cell suspension was filtered through a 0.4 μM cell filter and subjected to flow cytometric analysis to detect the amount of intracellular lipid ROS. The fluorescence intensities of cells per sample were determined by flow cytometry using the BD FACSAria cytometer (BD Biosciences). A minimum of 10,000 cells was analyzed for each sample. Data analysis was evaluated using the FlowJo Software.
10
Annexin V-FITC Apoptosis Analysis
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The percentages of viable (annexin-/propidium iodide [PI]-) or early apoptotic (annexin+/PI-) cells were quantified by using annexin V-fluorescein isothiocyanate (FITC) staining, which was used to detect phosphatidylserine that is externalized in the early phases of apoptosis. Annexin V is an important marker of early apoptosis in which changes in externalized phosphatidylserine levels occur prior to DNA fragmentation [14 (link)]. We used the Annexin V-FITC Apoptosis Analysis Kit according to the manufacturer’s instructions (Tianjin Sungene Biotech Co., Ltd., China). Mononuclear cells (1 × 105) were labeled, incubated in the dark for 15 min and immediately sorted by flowcytometry (BD FACSAriaTMш, USA). Marked annexin V-FITC staining (green) was analyzed using the FL-1channel, while PI staining (red) was assessed using the FL-2 channel. The data were analyzed by using the FlowJo software on a BD FACS Aria™ cytometer (BD BioSciences, USA).
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