In situ RNA hybridization with enriched kidney leukocytes (see above) was performed using the PrimeFlow RNA Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. In brief, single cell suspensions of kidney leukocytes were surface-stained in ice-cold PBS supplemented with 1% FCS, 5 mM EDTA and 0.02% NaN3, subsequently fixed and permeabilized. RNA targets were stained by incubation with gene-specific target probes, pre-amplification, amplification and hybridization with fluorescently labeled probes. Cells were acquired on a FACS LSR II Fortessa (BD Biosciences) or a Sony SP6800 Spectral Analyzer (Sony Biotechnology) and the data were analyzed with FlowJo software (Tristar) as described above, including pre-gating on viable and single cells for analysis.
Facs lsr 2 fortessa
Manufactured by BD
Sourced in United States
The FACS LSR II Fortessa is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It offers a range of excitation lasers and detection channels for sensitive and reliable data acquisition. The core function of the FACS LSR II Fortessa is to enable high-throughput, multi-parameter analysis of samples in a research or clinical laboratory setting.
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Lab products found in correlation
Primeflow rna assay kit, by Thermo Fisher Scientific (1 mentions)
Sp6800 spectral analyzer, by Sony (1 mentions)
Aqua viability dye, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by BD (1 mentions)
Ccr6 pe cy7, by BD (1 mentions)
Cd4 alexa700, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Ccr2 alexa 647, by BD (1 mentions)
Cxcr4 pe, by BD (1 mentions)
Cd33 apc, by BD (1 mentions)
Ccr7 fitc, by BD (1 mentions)
Cd209 fitc, by BD (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd14 percp cy5, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Hla dr percp cy5, by BD (1 mentions)
Cd8 apc cy7, by BD (1 mentions)
Celltrace violet, by BD (1 mentions)
8 protocols using facs lsr 2 fortessa
1
In Situ RNA Profiling of Kidney Leukocytes
2
Quantifying Cell Death by Flow Cytometry
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Cell death was quantified by the percentage of propidium iodide (PI; P4170, Sigma-Aldrich) positive staining cells detected by flow cytometric analysis. 2 × 105 cells per well were seeded in 6-well plates and treated with indicated drugs. After harvesting the cells, they were washed with phosphate-buffered saline (PBS) twice and then incubated with antibodies on ice for 30 min in the dark. Flow cytometric analysis was conducted on a FACS LSR II Fortessa (BD Biosciences) and the FACS data were analyzed with FlowJo software (Tree Star).
3
FcγR Expression in HBV Infection
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Multicolour flow cytometry was used to detect FcγRs on NK cells, B cells and monocytes in different phases of HVB infection. Briefly, six freshly obtained whole blood samples were added with six groups of fluorescent marker antibodies, including CD3‐PE‐Cy7, CD56‐APC, and CD16‐FITC; CD3‐PE‐Cy7, CD19‐APC, CD5‐PE and CD32‐FITC; CD3‐APC, CD4‐PE‐Cy7 and CD8‐PE; CD14‐PE and CD16‐FITC; CD14‐PE and CD32‐FITC; and CD14‐PE and CD64‐FITC. All antibodies were purchased from BD Biosciences (San Diego, CA, USA). After incubation in the dark for 30 minutes at room temperature, the freshly obtained whole blood was treated with RBC lysis buffer (BD Biosciences) for 3 minutes in the dark. Dead cells were excluded through staining with Aqua Viability Dye (Invitrogen). The samples were detected by FACS LSR II Fortessa (BD Biosciences) and analysed with FlowJo (Tree Star, Ashland, OR, USA).
4
Flow Cytometry Immunophenotyping Protocol
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Samples were acquired using a FACS LSRIIFortessa (BD Biosciences) and analysed using FlowJo (Tree Star, Ashland, OR, USA) and PESTLE and SPICE (National Institute of Allergy & Infectious Diseases, USA).
Compensation matrices were created on FlowJo using single stained anti-mouse Ig, κ/negative control compensation beads (BD Biosciences).
Compensation matrices were created on FlowJo using single stained anti-mouse Ig, κ/negative control compensation beads (BD Biosciences).
5
Multiparametric Lymphocyte Immunophenotyping
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Immunophenotyping of lymphocytes, monocytes, and monocyte-derived DCs was performed with the following antibodies: CD3-PerCP-Cy5.5 (clone: SK7), CD33-APC (clone: P67.6), and HLA-DR-PerCP-Cy5.5 (clone: G46.6), from BD Biosciences, CD209-FITC (clone: DCN46), CD8-APC-Cy7 (clone: SK1), CD80-PE (clone: L307.4), CD86-PE (clone: 2331-FUN-1), CCR7-FITC (clone: 3D12), CD14-PerCP-Cy5.5 (clone: M5E2), CD4-Alexa-700 (clone: RPA-T4), CXCR4-PE (clone: 12G5), CCR2-Alexa-647 (clone: 48607), CCR6-PE-Cy7 (clone: 11A9) from BD Pharmingen, CCR3-BV421 (clone: 5E8), CCR5-PE-CF594 (clone: 2D7/CCR5) from BD Horizon. FMO was used as a fluorescence background control. Briefly, 1 × 105 cells were incubated in the dark/room temperature for 30 min in the presence of antibodies at concentrations recommended by the manufacturers. We used FACS Lysing solution (BD Biosciences) for red cells lysing after staining of whole blood. Cells were then washed with buffered solution and at least 10,000 events were acquired using FACS LSRII FORTESSA (BD Biosciences).
Data were analyzed using the FACSDIVA (BD Biosciences) and/or the FlowJo software (Tree Star, Ashland, OR, USA). The gating strategy is described in FigureS2 in Supplementary Material.
Data were analyzed using the FACSDIVA (BD Biosciences) and/or the FlowJo software (Tree Star, Ashland, OR, USA). The gating strategy is described in Figure
6
Evaluating Dendritic Cells Migration via EVs
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Migration of iDCs (day 6) and mDCs (day 7) cultivated in the presence or absence of EVs derived SCC25 was evaluated using a transwell assay. iDCs or mDCs were collected and centrifuged (5 min/500×g) and stained using CellTrace Violet BV421 (Thermo Fisher Scientific) for 15 min at 37 °C, and then washed with PBS. Following the staining, cells were seeded on the upper chamber of a transwell (5.0 µM pores) in 24 well plates. DCs migration was induced using SCC25 cells cultivation in the lower chamber of the transwell, while for controls only cell medium was used in the lower chamber of the transwell. After 24 h the percentage of cells that migrated was evaluated by flow cytometry detecting CellTrace Violet stained DCs (iDCs and mDCs) and the antibodies CD11c- PE-CF594 (clone:B-ly6) from BD—Pharmingen and CD209—PerCP-Cy5.5 (clone:DCN46) from BD—Horizon. The events were acquired using FACS LSRII FORTESSA (BD Biosciences). The data analysis was performed using FlowJo software v.10.6.2 (BD Biosciences, https://www.flowjo.com ). The statistical analysis was performed using the GraphPad Prism software v. 7.02 (GraphPad Software, San Diego, CA, https://www.graphpad.com/scientific-software/prism ).
7
Comprehensive Phenotyping of Human PBMCs
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1.5 × 106 PBMC were stained for 30 min at 4 °C using antibodies against CD163 (BV421, clone GHI/61), CCR2 (BV510, clone K036C2), CD14 (BV605, clone MSE2), CD16 (APC-Fire750, clone 3G8), CD11b (BV711, clone ICRF44), HLA-DR (A488, clone L243), CD45 (PerCpCy5.5, clone HI30), CD209 (PE, clone 9E9A8), CD19 (PECy5, clone HIB19), CD3 (PECy5, clone UCHT1), CD56 (PECy5, clone 5.1H11), CD20 (PECy5, clone 2H7), TCRb (PECy5, clone UP26), and Fc block (clone FC1). All antibodies were purchased from BioLegend, except for Fc block which was obtained from BD Biosciences. Cell analysis was performed on a FACS LSRII Fortessa (BD Biosciences). Acquired data were analyzed using FlowJo software (Version 10.8.1, BD Biosciences).
8
B Cell Calcium Flux Assay
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Total HRO splenocytes at 107 cells/ml were loaded with 5 µM Indo-1 acetoxymethyl (I-1203; Thermo Fisher Scientific) in HBSS with 2% FCS for 30 min at 37°C. DAPI, anti–CD19-APC-Cy7, and anti–B220-PE were added for the last 15 min of incubation. Cells were washed twice and left at room temperature for 15 min in HBSS with 2% FCS. Cells were then equilibrated at 37°C, and signal at Indo blue and Indo violet of gated B cells was collected for 1 min to record baseline Ca2+ levels as the ratio of Indo violet/blue. Cells were then stimulated with anti-Igκ antibody, OVA conjugated with HA, OVA monomer, and OVA multimer, and Ca2+ levels were recorded for an additional 3 min. Ionomycin-stimulated cells were used as a positive control. Data were collected in a FACS LSR II Fortessa (BD) and analyzed using FlowJo.
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