The percentage of Treg and Th17 cells in spleen tissue were detected by flow cytometry. Spleen tissues were prepared into a single cell suspension. CD3 (BD, Cat#557666), CD4 (BD, Cat#552775), CD25 (BD, Cat#558642), IFN-γ (BD, Cat#557735), IL-4 (BD, Cat#562915) were surface staining, while Foxp3 (eBioscience, Cat#17-5773-82) and IL-17A (BD, Cat#564169) were stained after the cell membrane was destroyed by eBioscience Fix/Perm (Cat#00-5523-00) or BD Fix/Perm buffer kit (Cat#554714) respectively. FVS 780 (BD, Cat#565388) was used to identify the live or dead cells. LSR Fortessa cell analyzer (BD) and BD FACSDiva 8.0.3 software were used to detect the percentage of Th1, Th2, Treg or Th17 cells.
Fvs780
Manufactured by BD
Sourced in United States
The FVS780 is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It provides high-performance fluorescence detection and data acquisition capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Fix perm, by BD (4 mentions)
Lsrfortessa cell analyzer, by BD (4 mentions)
Il 17a, by BD (3 mentions)
Interleukin 4 (il 4), by BD (2 mentions)
Fix perm buffer kit, by BD (2 mentions)
Facsdiva 8, by BD (2 mentions)
Ifn γ, by BD (2 mentions)
Cocktail a, by BD (2 mentions)
Transcription factor buffer set, by BD (2 mentions)
Red blood cell lysis solution, by Miltenyi Biotec (2 mentions)
Fortessa lsr, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Cd4 buv496, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Facsdiva software version 8, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Roche (1 mentions)
Tnf α, by BioLegend (1 mentions)
Anti pd l1, by BioLegend (1 mentions)
31 protocols using fvs780
1
Quantification of Treg and Th17 Cells
2
Comprehensive Leukocyte Profiling in Follicular Fluid
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Single-cell suspensions of follicular and blood CD45-positive cells were obtained from FF or the peripheral blood of 5 women on the day of oocyte retrieval. Cell viability was assessed with the fixable viability stain 780 (FVS780). The anti-human antibodies used for surface staining of leukocytes are detailed in the supplemental data (Table S2 ). Cells were stained for 30 min at 4 °C. All antibodies and FVS780 were purchased from BD Biosciences (Franklin Lakes, NJ, USA). The BD LSRFORTESSA cell analyzer and the BD FACS Diva software version 8.0 (BD Biosciences) were used to perform flow cytometry. Flow cytometry standard files (FCS files) were further analyzed using FLOWJO version 10 software. The gating strategy used to determine the leukocyte subtypes is shown in the supplemental data (Fig. S1 ).
3
Single-Cell Profiling of Tumor Immune Composition
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Fresh tumors and organoids were prepared into single cell suspension after digestion in collagenase D (1mg/ml in DMEM, Roche) at 37°C for 30 min. The samples were stained for 20 min at room temperature with a comprehensive antibody staining panel as follows: CD3 (BD Bioscience, 560 910), CD4 (BD Bioscience, 561 005), CD8 (BD Bioscience, 561 952), CD45 (BD Bioscience, 564 106), CD69 (BD Bioscience, 565 155), FVS780 (BD Bioscience, 565 388). The cells were washed with staining buffer before phenotyping. A total of 100 000 cells were collected using the Beckman Coulter CytoFLEX and analyzed with FlowJo (Version 10.8.1, Treestar).
4
Cytokine-Induced Immune Marker Profiling
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A total of 1.5x104 cells per well were seeded in 96-well plates with medium containing either 100 ng/mL IFN-γ (Bio Legend, 570202) or 40 ng/mL TNF-α (Bio Legend, 570104) for two days. Cells were harvested and incubated with conjugated monoclonal anti-HLA-A2 (BioLegend, 343306, RRID:AB_1877227 ), anti-PD-L1 (Bio Legend, 329713, RRID:AB_10901164 ) and anti-ICAM-1 (BD, 559771, RRID:AB_398667 ) antibodies or respective isotype for 30 min at 4 °C. Nonspecific binding was blocked by using 1% Fc block. Cells were washed three times and analyzed by flow cytometry. Cell viability was determined using fixable viability stain FVS780 (BD, 565388, RRID:AB_2869673 ).
5
Immunophenotyping of Dissociated NMIBC Samples
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Frozen dissociated NMIBC samples obtained from 15 patients (CHU de Québec-Université Laval ethics approval #2012-253 and 2017-2749) were analyzed by flow cytometry. Cells were first incubated with human Fc block (#564219; BD Biosciences) for 15 min at 4 °C and then stained for 30 min at 4 °C in the dark with an antibody panel. Antibodies used are detailed in Supplementary Table 2.
For both human and murine cells, stained cells were analyzed using the BD LSRFortessa cytometer (BD Biosciences, San Jose, CA, USA). Data were processed using BD FACS Diva software (BD Biosciences), with analysis using FlowJo software (version 10.7.1; Flowjo, LLC, Ashland, OR, USA). Cell viability was assessed using a viability stain (FVS-780; BD Biosciences, or FVS-450; BD Biosciences), and doublets were excluded based on forward scatter-H against forward scatter-W. Compensation controls were done using compensation beads (BD CompBeads; BD Biosciences) and fluorescence minus one controls on fresh samples.
For both human and murine cells, stained cells were analyzed using the BD LSRFortessa cytometer (BD Biosciences, San Jose, CA, USA). Data were processed using BD FACS Diva software (BD Biosciences), with analysis using FlowJo software (version 10.7.1; Flowjo, LLC, Ashland, OR, USA). Cell viability was assessed using a viability stain (FVS-780; BD Biosciences, or FVS-450; BD Biosciences), and doublets were excluded based on forward scatter-H against forward scatter-W. Compensation controls were done using compensation beads (BD CompBeads; BD Biosciences) and fluorescence minus one controls on fresh samples.
6
T Cell Proliferation Assay with CFSE
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The splenocytes were isolated, mashing spleen with a plunger end of syringe into a 40-µm cell strainer. The erythrocytes were lysed using Easylyse (Dako). The cells were then labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE; Intercom). A 96-well round-bottom plate was precoated with an anti-CD3e (145-2C11, 5 µg/mL, eBioscience) for 2 h at 37 °C. The T cells (105 cells/well) were added and incubated for 72 h with an anti-CD28 antibody (37.51, 2.5 µg/mL, eBioscience) or complete media for control well in a humidified environment with 5% CO2 at 37 °C. After 3 d of culture, the cells were harvested and labeled with CD4 (GK1.5), CD8 (53-6.7) antibodies (BD Biosciences), and FVS780 (BD Biosciences) for cell viability. CFSE dilution was assessed by flow cytometry on a Fortessa X-20, and results were analyzed with ModFit LT software.
7
Multiparameter Immunophenotyping of Cells
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PE‐conjugated anti‐CD4 (RPA‐T4), anti‐CD16 (B73.1), APC‐conjugated anti‐CD8a (HIT8a), anti‐CD274 (PD‐L1) (29E.2A3), BV421‐conjugated anti‐CD3 (OKT3), anti‐CD14 (M5E2) and FITC‐conjugated anti‐CD279 (PD‐1) (EH12.2H7) were purchased from BioLegend Inc. FITC‐conjugated anti‐CD3 (HIT3a), BV421‐conjugated anti‐PD‐1 (EH12.1), BUV395‐conjugated anti‐CD56 (NCAM16.2) and FVS‐780 were purchased from BD Biosciences. FcR blocking reagent was purchased from Miltenyi Biotec B.V.& Co. KG.
8
Alveolar Lavage Fluid Collection
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After dissecting the mice, the trachea and lungs were exposed. After a trocar was inserted into the trachea, its needle was removed. The remaining hose was connected to a 1 mL syringe containing 800 µL PBS. Repeated suction was performed three times to recover the alveolar lavage fluid and cells, with more than 650 µL of liquid being recovered in each time. After centrifuged at 400 g for 5 min, the supernatant of the first suction was collected and frozen at -80 ℃ for detection of BALF protein concentration by a BCA Protein Assay Kit (Biyuntian) and inflammatory factor concentrations. The cell precipitates of all the suction medium were merged, in which the red blood cells were lysed by ACK lysing buffer (GBA1049201; corning). The cell precipitation was then divided into two parts. One part of the cells was used to count the total number of cells in the alveolar lavage fluid by a cell counting plate, and the other part cells were stained and analyzed by flow cytometry. The obtained cells were stained for FVS780 (565388; BD), CD11b (562950; BD), and Ly6G (127607; Biolegend) successively. CD11b and Ly6G were used to identify neutrophils. Data were acquired via CytoFLEX LX Flow Cytometer (Beckman Coulter, Inc.).
9
Th17 and Treg Cell Detection Protocol
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The spleen tissue was aseptically removed and prepared into a single-cell suspension. For Th17 (CD3+CD4+IL17A+) cells and Treg cells (CD3+CD4+ CD25 + FOXP3+) detection, the cells were stimulated by cocktail A for 4 h (BD, Cat# 550583). Samples were then washed and re-suspended in 1 × PBS stained with FVS 780 (BD, Cat#565388) to discriminate viable cells and then incubated with various surface markers. Consequently, samples were fixed by eBioscience Fix/Perm (Cat#00-5523-00) or BD Fix/Perm buffer kit (Cat#554714) to destroy the cell membrane and then were stained with FOXP3 (eBioscience, Cat#17-5773-82), IL-17A (BD, Cat# 564169). In the study, CD3 (BD, Cat# 557,666), CD4 (BD, Cat# 552,775) and CD25 (BD, Cat# 558642) were used. Finally, samples were analyzed with the LSR Fortessa cell analyzer (BD) and BD FACSDiva 8.0.3 software.
10
Phenotypic Characterization of Activated pDCs
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Referencing a previous in vitro study [10 (link)], PBMCs were stained with a fluorescent dye and fluorescent conjugated antibodies: BD Horizon Fixable Viability Stain 780 (FVS780) viability dye, PE-Cy7 Mouse Anti-Human CD123 (BD, 560826), BB515 Mouse Anti-Human Neuropilin-1 (CD304) (BD, 566036), and APC Mouse Anti-Human CD86 (BD, 555660) following the manufacturer’s instructions, and then fixed with 4% paraformaldehyde. PBMCs were cultured at 1 × 106 cells/mL in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% human AB serum (Sigma-Aldrich), 2 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific), and stimulated in the presence of 10 µg/mL TLR-7/8 agonist R848 (InvivoGen, San Diego, CA, USA) for 4 h. Afterward, the cells were harvested and stained with FVS780, PE-Cy7 Mouse Anti-Human CD123, BB515 Mouse Anti-Human Neuropilin-1 (CD304), and PE Mouse Anti-Human HLA-DR (BD, 556644) as described above. Data were collected using CytoFLEX (Beckman Coulter, Brea, CA, USA) and analyzed using the FlowJo version 10 software (BD). Isotype controls (BD) were used to check the background caused by nonspecific antibody binding. pDCs were defined as CD123+CD304+ subsets, and CD86 and HLA-DR were used as activation markers for pDCs.
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