CCA cells were harvested, fixed in 75% ethanol and stored at 4℃ overnight. Then, the cells were pre-treated and stained with Propidium (PI) & RNase Staining Buffer (SIGMA) for cell cycle analysis, while Annexin V-APC (Invitrogen) was used for apoptosis detection. The percentage of CCA cells at different stages and statuses were evaluated using flow cytometry (Novocyte D2060R, Agilent).
Novocyte d2060r
Manufactured by Agilent Technologies
Sourced in United States
The Novocyte D2060R is a flow cytometer designed for high-performance cell analysis. It features a compact design and provides reliable data acquisition and analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 apc, by Thermo Fisher Scientific (1 mentions)
Cd86 apc, by BioLegend (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Ifn γ pe, by BioLegend (1 mentions)
Il 2 apc, by BioLegend (1 mentions)
Apc il 17, by Thermo Fisher Scientific (1 mentions)
Cd19 pe cy7, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Percp cy5.5 cd8, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Macs miltenyi mouse tumour dissociation kit, by Miltenyi Biotec (1 mentions)
Magnetic bead, by Miltenyi Biotec (1 mentions)
Anti cd4 pe, by BD (1 mentions)
Anti cd19 pe, by BD (1 mentions)
Anti cd56 pe, by BD (1 mentions)
Anti cd16 apc, by BD (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Anti cd11c apc, by BD (1 mentions)
7 protocols using novocyte d2060r
1
Cell Cycle and Apoptosis Analysis
2
Mitochondrial and Oxidative Stress Assays
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Mitochondrial membrane potential assay kit with JC-1 (C2006, Beyotime, China) and reactive oxygen species assay kit (S0033S, Beyotime, China) were conducted for pre-treated hepatocytes according to the instructions as described previously (Zheng et al., 2021 (link)). Liver samples were separated, washed, ground, and filtrated to a single-cell suspension. About 1×106 cells were diluted into 100 ul phosphate buffered solution for subsequent staining. Antibodies against F4/80 FITC (123107), CD11b PE (101207), CD206 PE-CY7 (141719), and CD86 APC (105011) were obtained from BioLegend (California, United States), while 5 μl of each antibody (1:20) was added into samples and incubated for 1 h. Ad interim, fixation and permeabilization solution (554714, BD Biosciences, United States) was applied according to its manual. And finally, data acquisition was conducted on NovoCyte D2060R (Agilent, United States), and the analyses were performed by FlowJo v10.8 (BD Biosciences, United States).
3
Flow Cytometric Analysis of NK and DC Subsets
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Flow cytometry was used to detect the percentage of natural killer (NK) cells and dendritic cell (DC) subsets in the peripheral blood of mice. Peripheral blood was collected into tubes containing ethylenediaminetetraacetic acid. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood by density gradient centrifugation. PBMCs (1 × 106) were incubated with fluorescein isothiocyanate-conjugated rat anti-mouse antibodies against CD45/PE, NK1.1/APC, CD11C/PE-CY5, and CD3 for 60 min in the dark. The antibodies were purchased from Tonbo (Beijing, China). The cell samples were detected on Novocyte D2060R (Agilent, CA, USA), and the percentage of CD3–NK1.1+ NK cell subset and CD11C+NK1.1– DC subset was analyzed using NovoExpress 1.4.1.
4
Cell Cycle Analysis of CAL-62 Cells Treated with F1
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For the cell cycle analysis, 5 × 105 CAL-62 cells were seeded into 6-well plates and treated with different concentrations of F1 (0, 10, 20, 30, and 40 μg/mL) for 24 h and then collected and fixed in 75% ethanol at 4 °C overnight. The cells were incubated with PI/RNase Staining Buffer (BD Pharmingen, San Diego, CA, USA, 550825) at 37 °C for 30 min. The phase distribution of the cell cycle was assessed by a flow cytometer (Novocyte D2060R, Agilent, USA) equipped with NovoExpress software.
5
Multiparametric Flow Cytometry Analysis
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Anti-human mAbs included PE-Cy7-CD3 (Biolegend), PerCP-Cy5.5-CD8 (Biolegend), APC-IL-17 (eBioscience), PE-IFN-γ (Biolegend), APC-IL-2 (Biolegend), PE-Cy7-CD19 (Biolegend), FITC-Zombie (Biolegend), APC-fire750-CD69 (Biolegend), PerCP-Cy5.5-TGF-β (Biolegend), APC-IL-10 (Biolegend), PE-GrB (eBioscience). After stimulation, PBMC were harvested and first stained with surface antibodies followed by fixation/permeabilization (Cytofix/Cytoperm kit, BD Biosciences) and subsequent intracellular staining, as previously described [13 (link)].
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. Flow cytometry characterization of lymphocyte subsets is presented in Additional file1 : Figure S1.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. Flow cytometry characterization of lymphocyte subsets is presented in Additional file
6
Tumour Characterization and CAR T-cell Analysis
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Mice were sacrificed, and the tumours were collected, homogenised, and dissociated using the MACS Miltenyi Mouse Tumour Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purified CAR T cells were obtained by sorting (magnetic beads, Miltenyi Biotec) based on the manufacturer’s instructions. The phenotype of CAR T cell was detected via flow cytometry (ACEA Novocyte D2060R). Sorted CAR T cells were utilised for metabolism analysis and transmission electron microscopy.
7
Characterizing Lymphocyte Subsets in Liver Transplant Recipients
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The following reagents were all obtained from BD Biosciences: FITC-anti-CD3, CY5.5-anti-CD4, CY5.5-anti-CD8, PE-anti-CD19, APC-anti-CD16, PE-anti-CD56, PE-anti-CD4, FITC-anti-Lin1, PerCPCY5.5-anti-CD123, and APC-anti-CD11C. 5 mL of whole blood for ow cytometric measurement was taken from liver transplant recipients. Peripheral blood mononuclear cells (PBMC) were isolated by coll density gradient centrifugation and resuspended in phosphate-buffered saline (PBS). Then, PBMC were stained with antibodies mentioned above at 4℃ in the dark for 20 min. After that, PBMC were washed once with 2 mL PBS and resuspended in 400 µl PBS for ow cytometry analysis.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. The lymphocytes evaluated were T (CD3 + ), TCD4 (CD3 + CD4 + ), TCD8 (CD3 + CD8 + ), B (CD19 + ), NK (CD56 + CD16 + ), NKT (CD3 + CD56 + CD16 + ) and DC (lin1 -CD11c + and lin1 -CD123 + ). Flow cytometry characterization of circulating lymphocyte subsets was presented in Suppl Fig. 1.
The absolute numbers of lymphocyte subsets were calculated using the percentages obtained in ow cytometry and the lymphocyte counts obtained in routine blood tests on the same day.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. The lymphocytes evaluated were T (CD3 + ), TCD4 (CD3 + CD4 + ), TCD8 (CD3 + CD8 + ), B (CD19 + ), NK (CD56 + CD16 + ), NKT (CD3 + CD56 + CD16 + ) and DC (lin1 -CD11c + and lin1 -CD123 + ). Flow cytometry characterization of circulating lymphocyte subsets was presented in Suppl Fig. 1.
The absolute numbers of lymphocyte subsets were calculated using the percentages obtained in ow cytometry and the lymphocyte counts obtained in routine blood tests on the same day.
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