The Annexin V-FITC/PI apoptosis assay kit (Sungene Biotech, China) and flow cytometry were employed to analyze apoptosis. Annexin V has a high affinity for phosphatidylserine (PS), which is located in the cell membrane and becomes exposed in the early stage of apoptosis. Flow cytometry was performed with NovoExpress (ACEA Biosciences, USA) according to the ACEA NovoExpress Software Guide. Annexin V+PI– cells were those in the early stage of apoptosis, and Annexin V+PI+ cells were those in the late stage of apoptosis. In our study, the rate of cell apoptosis was represented as the percentage of early apoptotic + late apoptotic cells.
Novoexpress
Manufactured by Agilent Technologies
Sourced in United States, China
NovoExpress is a compact, benchtop flow cytometry system designed for routine cell analysis. It provides automated sample handling, data acquisition, and analysis capabilities to support a range of applications, including cell counting, viability assessment, and immunophenotyping.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte flow cytometer, by Agilent Technologies (14 mentions)
Novocyte, by Agilent Technologies (10 mentions)
Novocyte 3000, by Agilent Technologies (6 mentions)
Facscalibur, by BD (3 mentions)
Cd16 cd32, by Thermo Fisher Scientific (2 mentions)
Comp bead, by Thermo Fisher Scientific (2 mentions)
Quanteon flow cytometer, by Agilent Technologies (2 mentions)
Fcs express, by De Novo Software (2 mentions)
Cell wash, by BD (2 mentions)
Novocyte 1000, by Agilent Technologies (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Formaldehyde, by Avantor (2 mentions)
αcd16 32, by BioLegend (2 mentions)
Apc cy7 α cd19, by BD (2 mentions)
Alexafluor 647 α cd102 3c4, by BioLegend (2 mentions)
Redfluor 710 α cd11b, by Cytek Biosciences (2 mentions)
Fitc α cd11b, by BD (2 mentions)
Pe cy7 α cd19, by Cytek Biosciences (2 mentions)
Violetfluor450 α cd3, by Cytek Biosciences (2 mentions)
66 protocols using novoexpress
1
Annexin V-FITC/PI Apoptosis Assay
2
Multimodal Immune Cell Analysis
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Single-cell suspensions from the thymus, lymph node, spleen, lung, or liver were prepared and resuspended in staining buffer (PBS containing 1% FBS and 2 mM EDTA) and stained with the indicated antibodies (Abs). Cells were incubated with PMA plus ionomycin for 4 h, and GolgiStop (BD Bioscience, San Jose, CA, USA) was added 2 h before harvesting the cells to detect cytokines. To detect transcription factors (phospho-p65 and phospho-c-Jun), we followed the manufacturer’s protocol from eBioscience (San Diego, CA, USA). Data were acquired by NovoCyte (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed with NovoExpress (ACEA Biosciences Inc., San Diego, CA, USA).
3
Flow Cytometry Analysis of Immune Cells
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Cells were stained with mixtures of fluorescently labeled monoclonal antibodies (Abs) and analyzed by flow cytometry. Shortly, single-cell suspension was prepared from the LN by pressing tissue through a metal mesh after collagenase digestion (100 µg/ml, 10 min, 37 °C, Sigma-Aldrich, USA). Surface receptors were stained with selected antibodies (see Supplementary Table 1 .) and Zombie Red live/dead dye (Biolegend, USA) for 15 min at 4 °C in the dark. For IFNγ staining, cells were stimulated for 4 h (37 °C) in DMEM 10% FCS, and Cell Activation mixture (Cat#423304; BioLegend). Intracellular staining was done using Transcription factor buffer set (BD Biosciences, USA) according to manufacturer’s instructions. Stained cells were analyzed by flow cytometry (LSR Fortessa, BD Biosciences, USA; NovoCyte, Acea, USA) and analyzed with FlowJo (FlowJo LLC, USA) and NovoExpress (Acea, USA). For gating strategy of DC and macrophages and intracellular staining of T-cells for IFNγ, see Supplementary Figs. 4 , 5 .
4
Quantification of Peritoneal Immune Cells
5
Multiparametric Flow Cytometry Analysis
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For surface staining, cells were incubated with diverse combinations of fluorochrome-labeled antibodies against CD11c (clone N418), MHC class II (clone M5/114.15.2), CD4 (clone RM4-5), Ly6C (clone AL-21), CD103 (clone 2E7), CD11b (clone M1/70), Bst2 (clone eBio927), B220 (clone RA3-6B2), SIRP-α (clone P84), CCR9 (clone eBioCW1.2), or Siglec-H (clone eBio440c) for 15 min in the dark at 4°C as indicated. Finally, the cells were washed with Cell Wash (BD Biosciences). Appropriate isotype controls were used to define the threshold of negative versus positive staining. All antibodies were purchased from BD Biosciences or from eBioscience (Frankfurt am Main, Germany). Data were acquired using a FACSCalibur or FACSCanto II (BD Biosciences). Data analysis was performed using CellQuest Pro, FACSDiva Software (both BD Biosciences), or NovoExpress (ACEA Biosciences, San Diego, CA, USA).
6
Characterizing Cell Surface Markers
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Fluorescence-activated cell sorting analysis was used to determine cell surface marker expression. The protocol was performed as described by Iacobazzi et al. (2) (link). The following primary antibodies were used: 1:10 CD31-PE (Bio-Rad, Hercules, California), 1:600 CD44-APC (Thermo Fisher Scientific), 1:25 CD45-FITC (Bio-Rad), 1:10 CD73-APC (R and D Systems, Minneapolis, Minnesota), 1:20 CD90-PE (BioLegend, San Diego, California), and 1:5 CD105-PE (LSBio, Seattle, Washington). Analysis was performed on a NovoCyte flow cytometer (ACEA Bioscience, San Diego, California) using NovoExpress (ACEA Bioscience) for data collection and FlowJo (TreeStar, Ashland, Ohio) for analysis.
7
Cell Cycle Analysis of Cancer Cell Lines
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HCT-116 and SW620 cells were seeded in a six-well plate at an initial density of 2.4x105 cells/well and allowed to attach overnight in complete culture medium. Cells were divided into three treatment groups, as aforementioned in the apoptosis assay. Cells were starved in serum-free culture medium for 24 h, except that groups with NAC treatment were pretreated with 3 mM NAC for 2 h after starvation in serum-free medium for 22 h, followed by HT treatment for 12 h. Cells were harvested, washed twice with PBS, evaluated for cell cycle using the cell cycle assay kit (cat. no. BB-4104; BestBio) and analyzed using the Novocyte® flow cytometer with NovoExpress® version 1.2.4 software (ACEA Bioscience, Inc.).
8
Apoptosis Induction by δ-TT and Nec
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Cells were seeded in 6 cm Petri plate at a density of 1.5 × 105 cells/plate for 24 h and then treated for 48 h with δ-TT (20 μg/mL) alone or following a pre-treatment with Nec (50 μM) for 4 h. After treatment, the cells were harvested, washed with phosphate buffer solution (PBS), resuspended in binding buffer (BB) 1X and incubated with Annexin V-FITC/PI according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry Novocyte 3000 (Acea Bioscience, Inc., San Diego, CA, USA) and results analyzed by software Novo Express (Version 1.4.1). Each experiment was repeated three times.
9
Mitochondrial Membrane Potential Assay
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2 × 105 cells/well were seeded in a six-well plate and incubated overnight. The next day, various concentrations of PeME were treated for 24 h, and then cells were harvested with trypsin-EDTA (Welgene) and transferred into 1.5 ml microtubes (1 × 104 cell/tube). JC-1 dyes were diluted to a final concentration of 2.5 µg/ml. The cells were incubated for 15 min at 21‒23°C in the dark. After incubation, cells were washed with PBS, and resuspended in 500 µl PBS. Flow cytometry analysis was performed immediately. The solutions were divided by NovoCyte 1000 and visualized by NovoExpress (ACEA biosciences).
10
Flow Cytometry Analysis of Renal Inflammation
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To analyze renal inflammation, renal infiltrating inflammatory cells were extracted and analyzed using flow cytometry. In brief, one kidney from each SLN mouse underwent flow cytometry analysis. Kidneys were minced with a razor blade and digested with 1mg/ml collagenase type 4 (Cat.# LS004188, Worthington Biochemical Corp, NJ, USA) for 30 minutes at 37°C in a shaking incubator. After incubation, cells were immediately moved onto ice, and the digestion was stopped by adding RPMI media 1640 (Cat. # 12633, Thermo Fisher Scientific Inc, MA, USA). Red blood cells were lysed using RBC lysing buffer (Sigma). Cell clumps were disrupted using a syringe with 22G needle and then forced through a 70 µm filter. For flow cytometric analysis, cells were stained with antibodies against B220, CD3, CD4, CD8, CD11b, CD11c, CD21, CD23, CD25, CD45, CD69, CD80, CD86, F4/80, Gr1 (BD Biosciences, CA, USA). Approximately 50,000 events were acquired from each sample. All samples were run on a NovoCyte flow cytometer (ACEA Biosciences, CA, USA). Analysis was performed using Novoexpress (ACEA Biosciences, CA, USA). Experimental details are included in Supplementary Information
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