Adherent cells were digested in 2.5 g·L−1 trypsin (25200072; Invitrogen) and dissolved in 15 mL of staining buffer (phosphate‐buffered saline containing 0.2% BSA). After centrifugation at 300 g for 10 min, the cells were resuspended in 100 μL of staining buffer. Non‐specific binding was blocked with 5% BSA for 1 h, and the cells were then incubated with specific antibodies for 30 min at 4 °C, such as anti‐CD31/PECAM‐1 monoclonal antibody‐fluorescein isothiocyanate (1 µg·test−1, 11‐0311‐81; Invitrogen) and anti‐CD140b/PDGFR‐β monoclonal antibody‐PE (1 µg·test−1, 12‐1402‐81; Invitrogen). Cells were washed, dissolved, and then detected by flow cytometry (FC500 MPL; Beckman Coulter, Brea, CA, USA). Data acquisition and analysis were performed using an MXP Cytometer and cxp analysis , version 2.1 (Beckman Coulter).
Fc500 mpl
Manufactured by Beckman Coulter
Sourced in United States, Switzerland
The FC500 MPL is a flow cytometry system designed for clinical and research applications. It features high-performance optics, sensitive detectors, and advanced data analysis capabilities. The FC500 MPL is capable of analyzing a variety of sample types, including cells, microparticles, and other suspended particles.
Automatically generated - may contain errors
Lab products found in correlation
Anxvf 200t, by Immunostep (3 mentions)
Milliplex kit, by Merck Group (3 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fluorescein isothiocyanate apoptosis detection kit 1, by BD (2 mentions)
Pe bdca 1, by Miltenyi Biotec (2 mentions)
Pe bdca 2, by Miltenyi Biotec (2 mentions)
Pe bdca 3, by Miltenyi Biotec (2 mentions)
Cxp software, by Beckman Coulter (2 mentions)
Ficoll hypaque, by Cytiva (2 mentions)
P4170, by Merck Group (2 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (2 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Fitc conjugated secondary rabbit anti rat igg, by Vector Laboratories (1 mentions)
Winlist version 9, by Verity Software House (1 mentions)
H2dcfda, by Beyotime (1 mentions)
Dihydroethidium, by Thermo Fisher Scientific (1 mentions)
M36008, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dcfh da, by Beckman Coulter (1 mentions)
62 protocols using fc500 mpl
1
Identification of Endothelial and Pericyte Cells
2
Apoptosis Analysis by Flow Cytometry
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For the apoptosis experiment, following 48 hours of transfection, cells were cultured in 37℃ incubator for 24 hours. The fluorescein isothiocyanate Annexin V Apoptosis Detection kit I(BD Pharmingen, San Diego, CA, USA) was used. Briefly, the cells were collected and centrifuged at 2000 ×g for 5 min. Then the cells resuspended in 500 μl binding buffer, supplemented with 5 μl Annexin V and 5 μl propidium iodide (PI), for 15 min of dark treatment at the room temperature. The flow cytometry (FC500 MPL, Beckman Coulter, Brea, CA, USA) was used to analyze the samples.
3
Comprehensive MDSC Phenotyping Protocol
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All monoclonal antibodies used in the study were purchased from Biolegend. For MDSC identification, we used PE-Cy7-anti-HLA-DR, FITC-anti-CD14 APC-anti-CD33 and/or APC-anti-CD45 antibodies. For phenotyping MDSC, we first gated MDSCs using anti-HLA-DR/CD14 antibodies and then costained with PE-anti-CD11b, CD13, CD15, CD39, CD73, CD80, CD83, CD86, CD124 (IL-4Rα), PD-1 (CD279), PD-L1 (CD274), and PD-L2 (CD273). For intracellular cytokine staining, cells were surface stained with anti-CD4/CD8 antibodies followed by permeabilization with the Cytofix/Cytoperm kit (BD Biosciences) and then staining with PE-anti-IL-2 or PE-anti-IFN-γ antibodies. Isotype-matched antibodies were used as controls. Data acquisition and analysis were performed using the flow cytometer (FC500 MPL, Beckman Coulter) and FlowJo software (Tree Star, Ashland, OR) respectively.
4
DNA and Surface Labeling of P. falciparum IEs
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P. falciparum IEs were DNA labeled with ethidium bromide and surface labeled with rat anti-HB3VAR21-DBLβ_D4 (1:20) and fluorescein isothiocyanate (FITC)-conjugated secondary rabbit anti-rat IgG (1:150; Vector Labs) as described previously (50 (link)). Fluorescence data from ethidium bromide-positive cells were collected on an FC500 MPL flow cytometer (Beckman Coulter) and analyzed using WinList, version 9.0 (Verity Software House).
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5
Intracellular ROS Production Assay
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The intracellular ROS production levels were determined using a spectrofluorimetric method, H2DCFDA (Beyotime Institute of Biotechnology, Haimen, China) assay. R- and R+ cells were grown to a number of 107 and exposed to hypoxic conditions for 24 and 48 h. Cells were then incubated with DCHF-DA (20 mM) for 20 min at 37°C in a dark room. Subsequently, the cells were harvested in a trypsin-EDTA acid solution. Cell suspensions were centrifuged at 175 × g for 5 min at 37°C, then the supernatant was removed. The intensity of DCHF-DA fluorescence was measured and calculated by flow cytometry analysis (FC 500 MPL; Beckman Coulter, Inc.).
6
Mitochondrial Dynamics and Cell Death Analysis
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Cells were preloaded with 100 nM MitoTracker® Green FM (MTG, Thermo Fisher, M7514) and washed with phosphate-buffered saline (PBS) before treatment [19 (link)]. To monitor mitochondrial mass, cells were collected in FACS tubes and loaded with MTG, as previously reported [19 (link)]. For the assessment of cell death, cells were stained with annexin V-FITC (Immunostep, ANXVF-200T) for 15 min at 37 °C, and afterwards, propidium iodide (0.1 mg/mL) (PI, Sigma-Aldrich, P4170) was added to each tube to detect either the percentage of PI or annexin-positive (+) cells. Otherwise, we measured ROS levels by detecting the accumulation of superoxide with 5 μM dihydroethidium (Invitrogen, D1168). To study the mitochondrial membrane potential (MMP) and mitochondrial ROS, we used 20 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM, Invitrogen™, T668), and 2 μM MitoSOXTM (Invitrogen™, M36008), respectively. MitoSOX and dihydroethidium were oxidized to ethidium and emitted a red fluorescence. All stained cells were analyzed by flow cytometer (Beckman Coulter FC500-MPL).
7
Cisplatin-induced Cell Cycle Arrest Analysis
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Cells were seeded at a density of ~6×105 cells/ml and treated with 5 μmol/l cisplatin to determine the effects of hSulf-1 on cisplatin-induced cell cycle arrest for 24 h. Following incubation, cells were washed with PBS and fixed with 70% ethanol overnight at 4°C. Next, cells were stained with 1 ml propidium iodide (PI, Sigma-Aldrich) synthetic dye solution (20 μg/ml PI, 20 μg/ml RNase, 0.5% Triton X-100 and 1 g/ml sodium citrate) for 30 min at 37°C in the dark and then analyzed by flow cytometry using an FC 500 MPL instrument (Beckman Coulter, Miami, FL, USA). The cell number in each phase in every group was calculated using ModFit software (Verity Software House Corp., Topsham, ME, USA).
8
UV-Induced Apoptosis Quantification in HL-60 Cells
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HL-60 cell death was detected by flow cytometry (FC 500 MPL; Beckman Coulter Inc., Fullerton, CA, USA) using multicaspase assay kits (Guava Technologies, Burlingame, CA, USA). HL-60 cells were planted in a 24-well plate at a density of 1×106 cells/well and irradiated with UV LED at 0, 8, 15, 30 and 60 J/m2. Following incubation for 2 h at 37°C, the cells were harvested, washed with phosphate-buffered saline (PBS) and stained with sulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone (SR-VAD-FMK) and 7-amino-actinomycin D (7-AAD), according to the manufacturer's protocol. SR-VAD-FMK is a caspase inhibitor that covalently binds to multiple active caspases during apoptosis, and 7-AAD is a nucleotide stain that only stains cells when membrane integrity is compromised. A total of 5×103 cells per analysis were examined using flow cytometry. Unstained cells, cells stained with SR-VAD-FMK alone and cells stained with 7-AAD alone were used as controls to set up compensation and quadrants. SR-VAD-FMK positive/7-AAD negative cells (early apoptosis) and double positive cells (late apoptosis) were considered as the apoptotic cell population, while SR-VAD-FMK negative/7-AAD positive cells as the necrotic cell population.
9
Apoptosis Detection in Tumor Cells
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An apoptosis staining kit was used (BD Biosciences), following which tumor cell apoptosis was detected by flow cytometry. First, the tumor cells and supernatants were collected, following which they were centrifuged at 250 g for 5 minutes. The cells were then washed via PBS, and the washed cells were placed in a binding buffer and mixed with AnnexinV‐PE and 7AAD. Finally, the tumor cells were transferred to the flow tube and incubated at room temperature, in the dark, for 20 minutes. In the analysis stage, flow cytometry (FC500MPL, Beckman Coulter) was used for analysis.
10
Intracellular ROS Measurement in Hypoxic Fibroblasts
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Intracellular ROS production was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology, Shanghai, China). The fibroblasts were seeded in 60-mm culture dishes. The cells were then subjected to hypoxic conditions for 24 and 48 h and were then incubated with DCFH-DA (20 mM) for 20 min at 37°C in a dark place. Following incubation, the cells were harvested using trypsin-EDTA solution. The cell suspensions were then centrifuged for 5 min at room temperature and the supernatant was removed. The fluorescence intensity of DCFH-DA was measured and calculated using a flow cytometer (FC500 MPL; Beckman Coulter, Inc.).
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