GBM cells were transfected with miRNAs as described above in six well plates. Four days later, the cells were stained with FITC-Annexin V/PI Apoptosis Detection Kit (BD Pharmingen, USA) by following the manufacturer’s instruction, and analyzed by Beckman CytoFLEX Flow Cytometer (Beckman Coulter, USA). Annexin V-positive cells were considered to be apoptotic. For cell cycle analysis, the cells were fixed with 70% ethanol after 2 days from miRNA and stained with propidium iodide (PI) solution (Invitrogene, Thermo Fisher Sicentifics, USA) and RNase (Promega, USA) at room temperature. The PI-stained cells were subjected to flow cytometry by Beckman CytoFLEX Flow Cytometer (Beckman Coulter, USA). The obtained flow cytometry data was analyzed with FlowJo version 10 software (BD Biosciences, USA).
Beckman cytoflex flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Beckman CytoFLEX flow cytometer is an analytical instrument designed to perform multi-parameter analysis of cells and particles in suspension. It is capable of detecting and measuring various characteristics of cells, including size, granularity, and the expression of specific proteins or markers. The CytoFLEX utilizes a laser-based detection system to analyze the properties of individual cells or particles as they flow through the instrument.
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Lab products found in correlation
Fixation and permeabilization solution kit, by BD (4 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (2 mentions)
Protein transport inhibitor, by BD (2 mentions)
Pe anti human ifn γ antibody, by BioLegend (2 mentions)
Fitc anti human hla a2, by BioLegend (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Collagenase 8, by Merck Group (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Annexin 5 fitc pi apoptosis kit, by MultiSciences Biotech (1 mentions)
Anti cd86, by BioLegend (1 mentions)
Anti cd11c, by Thermo Fisher Scientific (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Pe cyanine7, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Apc cy7, by Thermo Fisher Scientific (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
36 protocols using beckman cytoflex flow cytometer
1
Apoptosis and Cell Cycle Analysis
2
Quantifying Apoptosis in Cell Lines
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All cells were seeded 24 h before treatment. At a confluence of about 10–15%, cells were treated with indicated drugs or diluents for 48 h, harvested and washed twice with PBS, permeabilized with 0.1% Triton X-100 and stained with propidium iodide. After 20,000 events were collected on a Beckman CytoFLEX flow cytometer, the percentage of sub-G1 events was quantitated using Beckman software. To compare apoptosis induced in different cell lines, 3–5 independent experiments were performed. The diluent-induced cell death was subtracted from each sample and drug-induced cell death was calculated using the formula: (deathobserved − deathcontrol)/(1 − deathcontrol) × 100%.
Alternatively, in combination assays, cells were harvested, washed with PBS, and stained with APC-labeled Annexin-V. Cells were then analyzed on a Beckman CytoFLEX flow cytometer. Three independent experiments were performed.
Alternatively, in combination assays, cells were harvested, washed with PBS, and stained with APC-labeled Annexin-V. Cells were then analyzed on a Beckman CytoFLEX flow cytometer. Three independent experiments were performed.
3
Cell Cycle Analysis of PC-3 Cells
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To assess cell cycle analysis, PC-3 cells were seeded in 6-well plates, at a density of 2.5 × 105 cells/well, and treated with vehicle (DMSO) or 10 μM of CUR or QRT, alone or in combination, for 24 h. Next, PC-3 cells were harvested after treatment by trypsinization, rinsed twice with PBS, pelleted by centrifugation, and fixed in 70% cold ethanol for 30 min at 4 °C. Then, samples were washed with PBS and stained using a solution containing 3.8 mM sodium citrate, 100 μg/mL RNAse, 50 μg/mL propidium iodide (PI), and 0.1% Igepal in PBS for 1 h at 37 °C. The samples were then analyzed using a CytoFLEX Beckman flow cytometer (Beckman Coulter Inc., Brea, CA, USA). All chemicals were from Sigma/Merck (Darmstadt, Germany).
4
Cell Cycle Analysis of PC-3 Cells
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In order to assess the cell cycle, PC-3 cells were seeded in well plates, at a density of 2.5 × 105 cells/well, and treated with vehicle alone or with 20 μM of CUR or DEX/CUR for 24 h. Flow cytometry was performed as described in [73 (link)]. Briefly, PC-3 cells were harvested after treatment with 0.025% trypsin for 3 min at 37 °C and then, after addition of 10% FBS in PBS, the cells were centrifuged, washed with PBS, and fixed in 70% cold ethanol. The harvesting of cells using these conditions led to high yield of undamaged cells. After fixation, cells were washed with PBS, treated for 15 min at 37 °C with RNase (100 μg mL−1) and then stained with PI (10 μg mL−1 in the dark for 30 min). The samples were then analyzed using a CytoFLEX Beckman flow cytometer (Beckman Coulter Inc., Brea, CA, USA).
All chemicals were from Sigma/Merck (Darmstadt, Germany).
All chemicals were from Sigma/Merck (Darmstadt, Germany).
5
Stellettin B-Induced Apoptosis Assay
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The following procedure was referenced from the literature [6 (link)]. OC2 and SSC4 cells were prepared at 2 × 105 cells per well in transparent 6-well plates (ThermoFisher, Rochester, NY, USA). Following overnight incubation, the cells were exposed to specified concentrations of stellettin B for 24 and 48 h. Next, the cells were washed twice with PBS, after which 1 µg/mL of AO reagent was added at 37 °C for 20 min. The cells were harvested and PBS-washed and finally diluted into 0.5 mL PBS. The Beckman CytoFLEX flow cytometer was used for evaluating the data from the AVO assay, using Beckman CytoExpert flow analysis software. A number of 10,000 cells per sample, or higher, was analyzed.
6
Quantifying Cellular Apoptosis via Annexin V/PI Staining
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Cell apoptosis was detected using an Alexa Fluor® 488 Annexin V/propidium iodide (PI) staining kit (Invitrogen; Thermo Fisher Scientific, Inc.). Cells from the different experimental groups were harvested, washed in ice-cold PBS, centrifuged at 100 × g for 5 min at 4°C, re-centrifuged using the same conditions, and resuspended in 400 µl 1X Annexin-binding buffer. Annexin V/PI staining solution was added to the mixture and cells were incubated in the dark for 15 min. Afterwards, 400 µl 1X Annexin-binding buffer was added to these mixtures and mixed gently. The green fluorescence of Annexin V (FL1) vs. red fluorescence of PI (FL2) was analyzed using a Beckman CytoFLEX flow cytometer (Beckman Coulter, Inc.) at 530 and 575 nm, respectively. At least 2×104 cells were examined per sample. Early apoptotic cells bound to Annexin V, but not to PI, whereas late apoptotic/necrotic cells displayed both types of binding, which was analyzed using WinMDI 2.8 software (The Scripps Research Institute).
7
Detailed Flow Cytometric Analysis of Lymphocytes
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Flow cytometric analysis was performed as described previously [43 ]. In brief, single-cell suspensions were isolated from spleens or MLNs, erythrocytes were removed by hypotonic lysis (Solarbio, #R1010), and dead cells were excluded by Fixable Viability Dye eFluor 780 (eBioscience) staining. For analysis of surface markers, cells were stained in PBS containing 2% bovine serum albumin (BSA) and 2 mmol/L EDTA. Single lymphocytes from the intestinal mucosa lamina propria were obtained by digestion with collagenase VIII (Sigma–Aldrich, Cat. C5138) and deoxyribonuclease I (Sigma–Aldrich, Cat. DN25) for 60 min, and the supernatants were then passed through a 70 μm cell strainer and subjected to Percoll (GE Healthcare) density gradient centrifugation. The FACS antibodies used are listed in Supplementary Table 3 . The stained cells were then analyzed with a Beckman CytoFlex flow cytometer (Beckman, USA).
8
Annexin V-based Apoptosis Assay
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Apoptosis was assayed using the annexin V–phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) or annexin V–adenomatous polyposis coli (APC)/7-AAD kit (Becton-Dickinson). The transfected cells were rinsed with ice-cold PBS and resuspended in 100 μL of 1 × binding buffer. Then, the liquid was stained with 5 μL 7-AAD and 5 μL annexin V–APC/PE and incubated for 15 min in the dark. Then, another 400 μL binding buffer was added into the mixture before cell apoptosis was detected on a Beckman cytoFLEX flow cytometer. The analysis of the above data was carried out using the CytExpert 2.3 software (Beckman Coulter, CA, USA).
9
Apoptosis Assessment by Flow Cytometry
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Cells were seeded into 6-well plates at a density of 5 × 105 cells/well and allowed to grow until reaching 50% confluence. Flow cytometry was used to evaluate cellular apoptosis. Apoptosis was measured by propidium iodide and annexin-V staining. Briefly, the cells were incubated for 15 min in darkness at 4°C with 5 μL of FITC-labeled recombinant annexin V (annexin V-FITC), followed by incubation for another 15 min with 5 μL of propidium iodide. Apoptosis was profiled using a Beckman CytoFLEX Flow Cytometer (Beckman Coulter, Suzhou, China).
10
Apoptotic Effect of miR-138 in GBM
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GBM cells were transfected with miR-138 mimics or miR-Ctrl as described above in 6 well plates in the presence of 10 µM TMZ or DMSO. After four days of transfection, the GBM cells were stained with FITC-Annexin V/PI Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s manual, and subjected to flow cytometry by Beckman CytoFLEX Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA). The obtained flow cytometry data was analyzed with FlowJo version 10 software (BD Biosciences, San Diego, CA, USA). Only Annexin V-positive cells were considered to be apoptotic cell populations.
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