Tumors were excised on day 15 after transplantation, cut into small pieces, and incubated in dissociation solution with 5 μg/mL collagenase type I (Sigma-Aldrich, St. Louis, MO, USA), 5 μg/mL hyaluronidase (Sigma-Aldrich), and 5 μg/mL DNase (Sigma-Aldrich). The solution was pipetted every 10 min during the incubation, and the suspension was dispersed through a 70 μm cell strainer. To identify cells from tumors, single-cell suspensions were stained with antibodies (BioLegend, San Diego, CA, USA). Live/dead cell discrimination was performed using Fixable Viability Stain 520 (Becton Dickinson and Company). Macrophages (CD45+ CD11b+ F4/80+ CD206+), monocytic-myeloid-derived suppressor cells (M-MDSCs, CD45+ CD11b+ ly6G- ly6Chigh), granulocytic-myeloid-derived suppressor cells (G-MDSCs, CD45+ CD11b+ ly6G+ ly6Clow), and T cells (CD45+ CD3+ CD4+ CD8+) were sorted using a BD FACSCanto II flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA).
Fixable viability stain 520
Manufactured by BD
Sourced in United States
Fixable Viability Stain 520 is a fluorescent dye used for the detection of live and dead cells in flow cytometry applications. The stain binds to intracellular proteins, allowing for the discrimination between viable and non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Aurora cytometer, by Cytek (1 mentions)
Software, by FlowJo (1 mentions)
Ab209775, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Igg from rat serum, by Merck Group (1 mentions)
Histofine simple stain mouse max po, by Nichirei Biosciences (1 mentions)
Purified rat anti mouse cd16 cd32, by BD (1 mentions)
Cytoflex flow cytometry, by Beckman Coulter (1 mentions)
Pharm lyse lysing solution, by BD (1 mentions)
Anti mouse cd4 pe, by BD (1 mentions)
Anti mouse cd8a apc, by BD (1 mentions)
Ultracomp ebeads compensation beads, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cell counting kit 8 reagent, by Dojindo Laboratories (1 mentions)
14 protocols using fixable viability stain 520
1
Tumor Dissociation and Cell Sorting
2
Quantifying Phosphorylated Histone H2A.X in ARPE-19 Cells
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ARPE-19 cells were seeded and treated in 12-well plates as previously described. After treatments, cells were collected and viable cells were stained with fixable viability stain 520 (564407, Becton Dickinson, Franklin Lakes, NJ, USA) for 15 min at RT in the dark. After washing, cells were permeabilized with glacial methanol for 10 min on ice. Nonspecific sites were blocked for 10 min at RT with a saturation buffer (PBS 1×, 1% BSA, and 0.2% Triton 100×), and then, cells were incubated with Alexa Fluor® 647 anti-p-histone H2A.X (Ser139) antibody (613408, BioLegend, San Diego, CA, USA) in the saturation buffer (1:100) for 30 min at RT. After washing, an analysis was performed using an Aurora cytometer (Cytek® Biosciences, Fremont, CA, USA) with Spectroflo® software (Biosciences, Torrance, CA, USA), and the data were analyzed using FlowJo v10 software (version 10, Tree Star, Ashland, OR, USA).
3
Cell Surface Staining of ILCs
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For cell surface staining of ILCs, freshly prepared human cells were stained with live/dead cell markers, Fixable Viability stain 520 (BD 564407), and Fc Block as for ILCs sorting. Cells were stained with surface antibodies against CD45, CD127, CD117, CRTH2, CD5, TIGIT and SLAMF1 and antibodies against same lineage markers for ILCs sorting for 30 min at room temperature. For each of the staining, paired samples from same patients were used. PBMC from more healthy donors was stained at same time. Cells were kept at 4°C and analyzed on a BD Symphony (BD Biosciences). Flow data was analyzed with FlowJo software (FlowJo LLC). Statistical analysis was performed by Mann-Whitney tests of unpaired nonparametric t tests or Kruskal-Wallis tests with Dunn’s multiple comparison tests. p-values were adjusted with the Benjamini-Hochberg method for multiple comparison tests. ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001, ∗∗∗∗ p-value < 0.0001.
4
Peptide-based T cell assay
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RNEU420-429 (PDSLRDLSVF), the immunodominant peptide of HER2/neu for MHC class I, and NP118-126 (RPQASGVYM), a peptide derived from a nuclear protein as a control, were obtained from Hokkaido System Science (Sapporo, Japan). An APC-anti-mouse CD8a monoclonal antibody (mAb), PE-anti-mouse IFN-γ mAb, and Fixable Viability Stain 520 were obtained from BD Biosciences (Franklin Lakes, NJ, USA). A therapeutic anti-PD-1 mAb (clone: RMP1-14) and anti-CTLA-4 mAb (clone: 9H10) were obtained from BioXcell (Lebanon, NH, USA). IgG from rat serum, administered as a therapeutic control, was obtained from Sigma-Aldrich (St. Louis, MO, USA). The antibodies (Abs) used for immunostaining were as follows: anti-CD8 mAb (ab209775, 1:2000, Abcam, Cambridge, United Kingdom), anti-CD4 mAb (ab183685, 1:1000, Abcam), anti-FOXP3 Ab (polyclonal, NB100-39002, 1:800, Novus Biologicals, Centennial, CO, USA), and Histofine Simple Stain Mouse MAX-PO (Nichirei Biosciences, Tokyo, Japan).
5
Tumor Immune Cell Profiling
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Tumor tissues were minced and digested in 1 mg/mL hyaluronidase, 1 mg/mL collagenase IV and 0.15 mg/mL DNase I for 2 hours and filtered with 70 μm strainer. Mononuclear cells were isolated with Ficoll-Paque PREMIUM 1.073 (Cytiva) as the manufacturer’s instruction, and blocked with Purified Rat Anti-Mouse CD16/CD32 (BD Pharmingen) for 1 hours at 4 oC. After staining with Fixable Viability Stain 520, anti-mouse CD3e PerCP-Cy5.5, anti-mouse CD8a APC and anti-mouse CD4 PE (BD Pharmingen) for 30 min at 4 °C, the cells were washed and analyzed by a CytoFLEX flow cytometry (BECKMAN COULTER). The spleens were grinded and filtered with 70 μm strainer, followed by lysing red blood cell using BD Pharm Lyse™ lysing solution. The spleen cells were collected, blocked, stained and analyzed as described above. The data were analyzed by FlowJo 10.6.2.
6
Quantifying Human Neutrophil LOX-1 Expression
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Human neutrophils were first blocked with FcR blocking reagent (Miltenyi Biotech, Germany) and then stained with Fixable Viability Stain 520 (1:4000 dilution, BD Biosciences, NJ, USA) and phycoerythrin (PE)-conjugated anti-LOX-1 antibody (clone 15C4, 1:800 dilution, BioLegend, CA, USA). UltraComp eBeads Compensation Beads (Thermo Fisher Scientific, MA, USA) were used for compensation. Flow cytometry data were acquired using BD Accuri C6 Flow Cytometer (BD Biosciences, NJ, USA) and analyzed by Flowjo v10.6.2 software (BD Biosciences, NJ, USA).
7
Quantifying BMSC Viability and HUVEC Proliferation
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Live/dead Staining: After incubating with material samples for 24 h, 3 and 7 days, BMSCs were washed with PBS twice, and then stained with calcein-AM and PI (Solarbio) for 30 min at room temperature according to the manufacturer's protocol.
The BMSCs cell viability test for 1, 3, 5 and 7 days was performed using cell titer-glo (Promega, USA) according to manufacturer's protocol and luminescence was measured using a chemiluminescence microplate reader (SpectraMax i3x, USA).
The cell viability staining on day 5 of co-culturing was determined using Fortessa flow cytometry (BD FACS, USA). BMSCs were digested with 0.25 % trypsin for 5 min at 37 °C, washed with PBS and then stained with fixable viability stain 520 (BD Bioscience) for 30 min according to manufacturer's protocol. Data were analyzed with FlowJo v10.8.1.
For cell proliferation assay, HUVECs (purchased from iCell Bioscience, China) were plated on prepared material samples in 96-well plates for 1, 3, 5 and 7 days in ECM media (Sciencell, USA). HUVECs were incubated with cell counting kit-8 reagent (Dojindo Laboratories, Japan) for 3 h and the OD value was determined using a spectrophotometer (BioTek, USA) at A450 nm.
The BMSCs cell viability test for 1, 3, 5 and 7 days was performed using cell titer-glo (Promega, USA) according to manufacturer's protocol and luminescence was measured using a chemiluminescence microplate reader (SpectraMax i3x, USA).
The cell viability staining on day 5 of co-culturing was determined using Fortessa flow cytometry (BD FACS, USA). BMSCs were digested with 0.25 % trypsin for 5 min at 37 °C, washed with PBS and then stained with fixable viability stain 520 (BD Bioscience) for 30 min according to manufacturer's protocol. Data were analyzed with FlowJo v10.8.1.
For cell proliferation assay, HUVECs (purchased from iCell Bioscience, China) were plated on prepared material samples in 96-well plates for 1, 3, 5 and 7 days in ECM media (Sciencell, USA). HUVECs were incubated with cell counting kit-8 reagent (Dojindo Laboratories, Japan) for 3 h and the OD value was determined using a spectrophotometer (BioTek, USA) at A450 nm.
8
Flow Cytometry Cell Viability Staining
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Cell suspensions were incubated with BD Horizon™ Fixable Viability Stain 450 (BD Biosciences, San Jose, CA, USA; Cat # 562247) at 4 °C for 30 min in the dark except the intestinal tissue and washed with ice-cold PBS. For the intestine, Fixable Viability Stain 520 (BD Biosciences; Cat # 564407) was used at 4 °C for 15 min in the dark. After adding anti-mouse CD16/CD32 (Thermo Fisher Scientific; Cat # 14-0161-85) to the samples, they were kept for 20 min in the fridge followed by a washing step. Subsequently, cells were stained with fluorochrome-conjugated anti-mouse antibodies at 4 °C for 30 min in the dark. Afterwards, FACS FlowTM (BD Biosciences; Cat # 342003) was added for washing.
9
Phenotypic Analysis of HIV-1 gp140-Specific B Cells
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To analyze the magnitude and phenotype of the HIV-1 gp140-specific B cell responses, 2 × 106 cells from the DLNs were seeded on 96-well plates, centrifuged and incubated with Fixable Viability Stain 520 (FVS 520; BD Biosciences). After blocking Fc receptors using anti-CD16/CD32 antibody (BD Biosciences), cells were incubated with 0.3 μg/106 cells of biotinylated clade C 96ZM651 gp140 protein (Biotin-XX Microscale Protein Labeling Kit; Invitrogen) for 30 min at 4°C in the dark. After washing, cells were incubated with the following fluorochrome-conjugated antibodies for surface markers: CD3-FITC, B220-PE-Cy7, IgD-APC-H7, CD38-PerCP-Cy5.5, IgG1-BV421, GL7-Alexa647, IgM-PE-CF594, and CD19-Alexa700 (all from BD Biosciences).
10
Flow Cytometry Analysis of Mouse Tumor Infiltrates
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Single-cell suspensions from mouse subcutaneous tumors were prepared as described above, counted, and resuspended in PBS at a concentration of 1×107 live cells/mL. One hundred microliters of each sample was plated for cell staining. Surface staining was performed at room temperature for 30 min, and intracellular staining was performed using a Foxp3-transcription factor staining kit (eBioscience). Live/dead cell discrimination was performed using Fixable Viability Stain 520 (BD Biosciences). Anti-mouse antibodies against the following antigens were used for flow cytometry: CD3 PerCP-CY5.5 (17A2, BioLegend, 1:100), CD45 PerCP (30-F11, BioLegend, 1:200), NK1.1 APC (PK136, BD Biosciences, 1:50), Foxp3 PE (MF23, BD Biosciences, 1:100), Ly6G PE (1A8, BioLegend, 1:100), Ly6C APC (HK1.4, BioLegend, 1:100), CD11b BV421 (M1/70, BioLegend, 1:100), CD11b PerCP-CY5.5 (HL3, BioLegend, 1:100), F4/80 APC (T45–2342, BD Biosciences, 1:50), CD25APC (3C7, BioLegend, 1:100), CD4 APC (GK1.5, BioLegend, 1:100), and CD8a PE (53-6.7; BioLegend, 1:100). All flow cytometry analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).
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