Were performed as described [39 (link)]. Briefly, the pEGFP-Pem1 plasmid was digested with HindIII or I-SceI for 8–12 h to generate free DNA ends. pCherry plasmids were co-transfected with linearized DNA to control for transfection efficiency. shScr and shk-h cells were transfected at ~20–25% confluency and allowed to grow for three days. Transfections were performed using Lipofectamine-2000 using the manufacturer’s instructions. Flow cytometric analyses were performed using a Beckman-Coulter Cytomic FC 500 flow cytometer.
Cytomic fc500
Manufactured by Beckman Coulter
Sourced in United States
The Cytomic FC500 is a flow cytometer designed for research applications. It is capable of detecting and analyzing multiple parameters of individual cells or particles suspended in a fluid stream. The instrument uses laser-based technology to provide quantitative and qualitative data on the physical and biological characteristics of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Nile red, by Beckman Coulter (2 mentions)
Nile red, by Merck Group (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Cytexpert, by Beckman Coulter (2 mentions)
Multicycle for windows, by Phoenix Pharmaceuticals (2 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Apoptosis detection kit, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Cd11a, by BioLegend (1 mentions)
Golgiplug, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Binding buffer, by BD (1 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions)
Cxp analysis, by Beckman Coulter (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Mitoprobe jc 1 assay kit, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
Propidium iodide, by BD (1 mentions)
26 protocols using cytomic fc500
1
Plasmid Transfection and Flow Cytometry
2
Nile Red Lipid Staining of Cardiomyocytes
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Nile red (Sigma Aldrich) was used to stain lipids. Rat ventricular cardiomyocytes were plated in each well of 4‐well plates and allowed to attach for 24 hours. After being paced for 24 hours, the cells were harvested through trypsinization, washed in PBS, and suspended in 5 μg/mL Nile red. After incubation for 15 minutes at 40°C, the cells were washed 3 times and suspended in PBS. To determine the intracellular Nile red content using flow cytometry, 10 000 cells per sample were analyzed using the Cytomic FC500 (Beckman Coulter, Indiana). The fraction of RV‐40 ventricular myocytes expressing Nile red was determined in 10 000 sorted myocytes. Nile red fluorescence was collected through a 530±30 nm wavelength.
3
Apoptosis Quantification in H9c2 Cells
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The H9c2 cells were isolated from the co-cultures using a MoFlo XDP high-speed flow cytometry sorter (Beckman Coulter Brea, CA, USA). Cell apoptosis was measured using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) with an apoptosis detection kit (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's protocol. The samples were analyzed on a fluorescence activated cell sorter (Cytomic FC500; Beckman Coulter) within 1 h. The numbers of apoptotic cells, including Annexin V-positive/PI-negative and double-positive cells, were counted and expressed as a percentage of the total cell count.
4
Annexin V-FITC Apoptosis Assay
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The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptosis. Briefly, the cells were trypsinized and washed twice with ice-cold phosphate-buffered saline. The cell pellet was resuspended and incubated in Annexin V binding buffer containing fluorescein isothiocyanate-conjugated Annexin V (1 μg/mL) and propidium iodide (50 μg/mL) for 15 min at room temperature. Analyses were performed using a Cytomic FC500 flow cytometer (Beckman Coulter, Brea, CA, USA).
5
Exploring Immune Profiles in Kidney Cancer
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Fifteen fresh tumor and adjacent normal kidney samples were resected from our institute to evaluate the levels of CCL5+ TAMs infiltration, the immune stimulatory molecules of CD8+ T cells, including TNF-α, IFN-γ, PRF-1, GZMB, and CD69, as well as the signatures of exhausted features in CD8+ T cells, including PD-L1, TIM-3, TIGIT, CTLA-4, and LAG-3. The relevant information regarding the antibodies utilized in these experiments is summarized in Supplementary Table 2
6
Phenotypic Analysis of NK Cells
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The expression of cell surface-bound receptors and intracellular proteins of NK cells was assayed by flow cytometry, with the commercially available antibodies against 2B4, granzyme B, IFN-γ, NKG2D, NKp30, NKp44, NKp46, perforin, Siglec-7 (eBioscience, San Diego, CA, USA), CD11a, CD18, ILT2 (BioLegend, San Diego, CA, USA), Siglec-9, and TNF-α (BD Biosciences, San Joes, CA, USA) with fluorescence-conjugation. For intracellular staining of cytokines, GolgiPlug (BD Biosciences) was added to block cytokine secretion. Samples were subjected to Cytomic FC500 or CytoFLEX (Beckman Coulter, Fullerton, CA, USA) flow cytometry equipment and results were analyzed using FlowJo software (FlowJo v10, LLC, Portland, OR, USA) or CytExpert (CytExpert 2.1, Beckman Coulter).
7
Cell Cycle Analysis of RPMI-8226 Cells
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RPMI-8226 cells were prepared at a concentration of 5.0×105 cells/mL and then seeded at a density of 1.0×106 cells/well in 6-well cell culture plates, treated with the indicated concentrations of GO, DOX, and GO/DOX (Fourier transform infrared spectrum of GO/DOX confirmed the effective loading of DOX on GO),18 and cultured for 24 hours at 37°C in a 5% CO2 humidified incubator. Cells were harvested by centrifugation at 1,000×g for 5 minutes at room temperature, washed twice with ice-cold phosphate buffered saline (PBS), and fixed with 70% ethanol at 4°C overnight. The fixed cells were suspended in PBS and further treated with propidium iodide (PI) for 30 minutes at 37°C in the dark. The cells were then centrifuged at 1,000×g, and the number of cells at the different phases of the cell cycle was analyzed using flow cytometry (Cytomic™ FC500; Beckman Coulter, Miami, FL, USA) equipped with the SYSTEM II™ analysis software (Multicycle AV for Windows). In cell-cycle analysis, the RPMI-8226 cells were gated on linear forward scatter and linear side scatter in order to remove fragments or impurities.
8
Comprehensive Immune Cell Profiling
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Fc receptors were blocked with mouse Fc block and the dead cells were detected using Fixable Viability Dye eFlour™ 506 or 510 (eBioscience) before cell surface staining. For Breg staining, CD19-FITC or PE/Cy5, CD24-PE, CD38-PE/Cy7, CD1d-PE, and CD5-FITC mAbs were used. For intracellular IL-10 staining, cells were stained with CD19-PE/Cy5 or APC mAbs. Cells were washed, fixed with IC Fixation Buffer (eBioscience), permeabilized with Permeabilization Buffer (eBioscience), and stained with IL-10-PE. For Treg staining, cells were stained with combinations of CD4-FITC and CD25-PE/Cy5.5 or APC mAbs, fixed and permeabilized with Fixation/Permeabilization solution (eBioscience) and Permeabilization Buffer, and stained for detection of intracellular Foxp3-PE mAbs. For apoptotic cell detection, cells were washed twice with cold PBS and then resuspended in 1× Binding Buffer (BD Biosciences), and then the cells were stained with CD4-FITC, APC Annexin-V, and 7-AAD and incubated for 15 min at RT in the dark. Last, 400 μl of 1× Binding Buffer was added. Data were acquired using Cytomic FC500 or Cytoflex (Beckman Coulter) and analyzed using CXP Analysis and Cytexpert (Beckman Coulter).
9
Mitochondrial Membrane Potential Monitoring during Apoptosis
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The mitochondrial membrane potential (∆ψm) during apoptosis was monitored by a MitoProbe JC-1 assay kit (Molecular Probe), which is a lipophilic cationic dye. In brief, HSC-T6 cells were exposed to SSd (1 μM) for 30 and 60 min, and the cells were subsequently labeled with 2 μM JC-1 dye for 30 min at 37 °C. All cells were collected, washed twice with PBS, and analyzed by flow cytometry (Cytomic FC 500, BECKMAN COULTER). For mitochondrial staining, HSC-T6 cells were grown on coverslips for 16 h. After treatment with 1 μM SSd for 0, 15, 30 and 60 min, mitochondria were stained with 100nM MitoTracker® Deep Red FM (Invitrogen™, Life Technologies) for 30 min at 37 °C. DAPI (Molecular Probe) was adopted as a nuclear counterstain, and images were acquired by a confocal laser-scanning microscope (TCS SP5, LEICA).
10
Nile Red Staining of Stretched Atrial Myocytes
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Nile red (Sigma Aldrich, St. Louis, MO, USA) was used to stain intracellular lipid. The HL-1 atrial myocytes were plated on silicone rubber culture dishes and cells were allowed to attach for 48 h. After 8 h of stretching, the cells were harvested by trypsinization, washed in PBS, and suspended in 5 μg/mL of Nile red. After incubation for 15 min at 40 °C, the cells were washed three times and suspended in PBS. In flow cytometry of intracellular Nile red content, 10,000 cells per sample were analyzed with a Cytomic FC500 (Beckman coulter, Indianapolis, IN, USA). The fraction of HL-1 atrial myocytes expressing Nile red was determined in the sorted 10,000 myocytes. Nile red fluorescence was analyzed at a wavelength of 530 ± 30 nm. A total of three experiments, comparing stretched and non-stretched myocytes, were performed. Each experiment was performed six times in each group.
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