Following treatment with RBCEVs, cells were collected and washed with PBS. Apoptosis was determined by Annexin V (ANXV)/propidium iodide (PI) staining with the apoptosis detection kit (Life Technologies). Briefly, 50,000 treated cells were incubated with ANXV and PI in binding buffer for 15 min at 4°C. The cells were then analyzed by FACS LSRII cytometer (BD BioSciences, USA) or CytoFlexS cytometer (Beckman Coulter). Flow cytometric plots were generated using FlowJo v10.0.7.
Facs lsrii cytometer
Manufactured by BD
Sourced in United States
The BD FACS LSRII cytometer is a flow cytometry instrument used for the analysis of cells and particles. It is designed to detect and measure various characteristics of cells, such as size, granularity, and the presence of specific markers or proteins on the cell surface. The FACS LSRII cytometer utilizes multiple lasers and detectors to provide high-resolution data on the analyzed samples.
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Lab products found in correlation
Facsdiva software, by BD (4 mentions)
Apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Cytoflex s cytometer, by Beckman Coulter (1 mentions)
Prism 8, by GraphPad (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
123count ebeads, by Thermo Fisher Scientific (1 mentions)
Fc block, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Lsrii flow cytometer, by Tree Star (1 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Tamra, by Merck Group (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti insulin, by Agilent Technologies (1 mentions)
Accumax, by STEMCELL (1 mentions)
Facscalibur, by BD (1 mentions)
Anti thy1, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
14 protocols using facs lsrii cytometer
1
Apoptosis Evaluation by ANXV/PI Staining
2
Tetramer-based Enrichment of Antigen-specific T Cells
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2 × 108 freshly isolated PBMCs were incubated with a mix containing 100 μL of AAV capsid-specific PE-conjugated tetramers (20 μg/mL) and 100 μL of irrelevant APC-conjugated tetramers (20 μg/mL). After washes, tetramer-stained cells were enriched using anti-PE antibody-coated immunomagnetic beads on LS columns (Miltenyi Biotech) according to manufactured instructions. After enrichment, cells were stained with an antibody panel that is summarized in Table S2 . Counting beads (123count eBeads, eBioscience) were used to normalize results. Cells were acquired on a FACS Fortessa X20 cytometer, a FACS LSR II cytometer, or a FACS Aria III sorter (BD Biosciences) and analyzed with FlowJo software (version 10, Tree Star Inc.). For PE-labeled Tetramer+ CD8+ T cell detection and phenotyping, CD3+ CD8+ CD4− T cells were gated on single live dump− (CD14− CD16− CD19−) cells. CD45RA, CD45RO, and CCR7 expression was then assessed on PE+ APC− CD8+ T cells. Statistical analyses were performed using GraphPad Prism 8.0.1. Results were considered non-significant when p > 0.05.
3
Immunophenotyping and Cytokine Analysis
4
Quantifying Intracellular ROS Levels
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To monitor reactive oxygen species (ROS) formation in response to a treatment with IFNβ1 or 4μ8C, the ROS was labeled using 2′-7′-dichlorofluorescin diacetate (Sigma). Cells were washed in Hank's balanced salt solution buffer, stained for 30 min with 10 μM 2′-7′-dichlorofluorescin diacetate in 0.5 ml fetal calf serum free medium at 37°C, washed twice in Hank's balanced salt solution, trypsinized, resuspended in cell-culture media, and measured. The fluorescence of the intracellular oxidized product dichlorofluorescein was measured by flow cytometry using a FACS LSR II cytometer (BD Bioscience).
5
Peptide Uptake in DRG Neurons and Cell Lines
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DRG neurons expressing YFP under the Thy1 promoter (Feng et al, 2000 (link)) were cultured on a 4 Well Glass‐Bottom slide chamber (µ‐Slide, ibidi 80427) for 24 h prior to treatment. For cold treatment (4°C), plates were taken out of the incubator and onto ice and taken in to the cold room (2‐8°C) to cool for 20 min. Then, medium was replaced with cold (4°C) growth medium supplemented with 5 μM R4 (FGGRGRGGFGGRGGFRGG) or N4 (FGGNGNGGFGGNGGFNGG) peptides N‐terminally conjugated with TAMRA (Sigma). Following additional 30 min, cells were washed three times in cold PBS, fixed in cold 4% PFA for 20 min, and imaged in PBS using Olympus FV1000 Confocal laser‐scanning microscope with a 60× water immersion objective (UPLSAPO 60× O NA:1.35). Average TAMRA intensity per pixel was measured using FIJI software. Cells were traced manually based on the phase contrast channel.
For analysis of peptide uptake into HEK‐293 and U937 cells, 5 μM TAMRA‐conjugated peptides were added to the growth medium for 60 min. Then, cells were collected into 15‐ml tubes for washing with cold PBS in pre‐cooled centrifugation (2 × 2 min, 1,000 × g). Cells then were suspended in 400 µl cold PBS and filtered through a nylon mesh into 5‐ml tube to obtain a uniform single‐cell suspension. Cells were analyzed on a FACS‐LSRII cytometer (BD Biosciences) using BD FACSDIVA software (BD Biosciences).
For analysis of peptide uptake into HEK‐293 and U937 cells, 5 μM TAMRA‐conjugated peptides were added to the growth medium for 60 min. Then, cells were collected into 15‐ml tubes for washing with cold PBS in pre‐cooled centrifugation (2 × 2 min, 1,000 × g). Cells then were suspended in 400 µl cold PBS and filtered through a nylon mesh into 5‐ml tube to obtain a uniform single‐cell suspension. Cells were analyzed on a FACS‐LSRII cytometer (BD Biosciences) using BD FACSDIVA software (BD Biosciences).
6
Insulin-positive cell isolation and analysis
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Isolated islets from mice dosed with AAV8-INS-Grx1-roGFP2 were dispersed using 1 ml of Accumax (StemCell Technologies) containing 2 U/mL of DNAse I for 10 min at 37 °C. Single cells were fixed and stained with anti-insulin (1/10 dilution, Dako) for 90 min at room temperature. Cells were washed and incubated with wash buffer containing secondary antibody (Alexa-fluor 568, 1/500 dilution, Invitrogen) for 30 min, room temperature. After the staining, the cells were washed, filtered and acquired on a FACS LSR II cytometer (BD). The cells were analyzed using FlowJo software (v 7.6.5, Tree Star). Initially, the cells were gated on FSC-A and FSC-H for doublet exclusion, followed by a strategy to gate on FSC (size) and SSC (granularity) to select a population compatible with β cells, within which the insulin-positive cells were evaluated, and finally GFP positivity of insulin-positive cells.
7
Flow Cytometric Analysis of Activated T Cells
8
Characterizing RBCEV Uptake in Myotubes
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The differentiated C2C12 myotubes in a 24-well plate were pre-treated with 10 μM wortmannin or 0.5 μM cytochalasin D for 1 h followed by incubation with 0.2 μg/μL CFSE-RBCEVs or MG640-RBCEVs for 6–24 h in a humidified incubator at 37°C with 5% CO2. For immunofluorescent staining, the cells were rinsed twice with PBS and fixed for 10 min using 4% paraformaldehyde (Alfa Aesar) in PBS. Blocking buffer (5% donkey serum and 0.1% Triton X-100 in PBS) was applied for 1 h at room temperature. After incubation with mouse anti-human GPA antibody (BioLegend) for 2 h at 4°C, the cells were incubated with donkey anti-mouse secondary antibody conjugated with Alex Fluor 594 fluorophore (Jackson ImmunoResearch) for 1 h at room temperature. The cells were then rinsed in PBS and stained with Hoechst 33342 (Abcam) and MemGlow 640 for 10 min at room temperature. The cells were subsequently washed three times with PBS. Images were acquired using an Olympus FV3000 confocal microscope (Olympus, Japan). Image acquisition was conducted using FluoView software. For flow cytometry analysis, the cells were harvested and washed with PBS twice and re-suspended in FACS buffer. The data were analyzed using a FACS LSRII cytometer (BD BioSciences). Flow cytometric plots were generated using FlowJo v.10.0.7.
9
Assessing MDSC-mediated T-cell Suppression
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To evaluate the ability of CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSC) isolated from the spleen from L2- IL-1β transgenic mice to suppress antigen specific T cell proliferation, we performed a T-cell proliferation assay. A single cell suspension of splenocytes isolated from wild-type mice was made by homogenizing spleen with a 1 ml syringe through a 70 μM filter into a 50 ml conical tube. Red blood cells were lysed using lysis solution and quenched with HBSS. Red blood cell depleted murine splenocytes were resuspended at 1 × 106/ml and labeled with 5 μM green fluorochrome carboxyfluorescein succinimidyl ester (CFSE). Cells were activated with anti-CD3/CD28 coated beads (Gibco, Life technologies) and seeded in triplicate and cultured in RPMI-1640 supplemented with 10% fetal bovine serum. Splenocytes were cultured either alone or in the presence of CD11b+Gr-1+ cells isolated from L2-IL-1β mice at different ratios 0:1, 1:1, 1:2, 1:4, 1:8, 1:16. After 72 h, cells were collected and stained with 7AAD, anti-CD45, CD3, CD4, Gr-1 and CD11b mAbs cocktail. Proliferation was determined by CFSE dilution and flow cytometric analysis on a FACSLSRII cytometer (BD Biosciences) with initial gating on the CD3+/CD4+ populations.
10
Immunophenotyping of Dendritic Cells
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