Intracellular ROS levels were also measured using a ROS detection assay kit (cell-based ab139476). Cells were cultured in 6-well plates and exposed to high glucose and EMPA treatment for 7 days. According to the manufacturer's instructions, cells were collected, washed with 1X wash buffer, and centrifugated for 5 min at 400 × g. According to the kit protocol suggestion, 1 × 105 cells were stained with detection reagent for 1 h at 37 °C in the dark with periodic shaking. Measurements were carried out using BD Accuri C6 Plus Personal Flow Cytometer (BD biosciences) at Ex/Em = 490/525 nm. Data processing was performed using FlowJo BD Accuri C6 Plus software for windows.
Accuri c6 plus personal flow cytometer
Manufactured by BD
Sourced in United States
The BD Accuri C6 Plus Personal Flow Cytometer is a compact, benchtop flow cytometer designed for analysis of cells and particles. It is capable of measuring multiple parameters, including size, granularity, and fluorescence, on individual cells or particles within a sample.
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Lab products found in correlation
Annexin 5 fitc apoptosis staining detection kit, by Abcam (1 mentions)
Fitc annexin 5 apoptosis detection kit with 7 aad, by BioLegend (1 mentions)
Cycletest plus dna kit, by BD (1 mentions)
Autophagy assay kit, by Abcam (1 mentions)
Dihydrorhodamine, by Merck Group (1 mentions)
Accuri c6 plus, by BD (1 mentions)
9 protocols using accuri c6 plus personal flow cytometer
1
Intracellular ROS Quantification
2
Annexin V-FITC Apoptosis Assay
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Cell death was evaluated using an annexin V-FITC apoptosis staining/detection Kit (ab273273, abcam). Cells were cultured in 6-well plates and exposed to HG and tirzepatide treatment for 7 days. According to the manufacturer's instructions, cells were collected with trypsin, washed with 1X wash buffer, and centrifuged for 5 min at 400 ×g. According to the kit protocol suggestion, 1 × 105 cells were resuspended in 500 μL of 1X Binding Buffer, then 5 μl of Annexin V-FITC and 1 μl of SYTOX Green dye added to each sample and for 10 min incubated at room temperature in the dark. Measurements were carried out using BD Accuri C6 Plus Personal Flow Cytometer (BD biosciences) at Ex/Em = 480/530 nm. Data processing was performed using FlowJo BD Accuri C6 Plus software for windows.
3
Apoptosis and Cell Cycle Analysis
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Apoptotic cells were detected using FITC Annexin V Apoptosis Detection Kit with 7-AAD (640922, BioLegend, San Diego, CA). Cells were washed twice with cold BioLegend’s Cell Staining Buffer and then resuspended in Annexin V Binding Buffer. For each staining reaction, 105 cells in 100 μL were incubated with 5 µL of FITC Annexin V, 5 µL of 7-AAD Viability Staining Solution or both for 15 minutes at room temperature in the dark before the addition of 400 µL Annexin V Binding Buffer to each tube.
Cell cycle was detected by flow cytometry with BD Cycletest™Plus DNA kit (340,242, BD Biosciences, Franklin Lakes, NJ) as described previously.21 (link) Briefly, cells were harvested at 72 hours after knockdown, washed three times with buffer solution, and adjusted to a concentration of 106 cells/mL. Subsequently, 5×105 cells were incubated with solution A (Trypsin buffer), solution B (Trypsin inhibitor and RNase buffer), and Solution C (PI stain solution) for 10 minutes respectively in the darkroom before analysis. All flow cytometry analyses were performed using BD Accuri™ C6 Plus personal flow cytometer (BD Biosciences).
Cell cycle was detected by flow cytometry with BD Cycletest™Plus DNA kit (340,242, BD Biosciences, Franklin Lakes, NJ) as described previously.21 (link) Briefly, cells were harvested at 72 hours after knockdown, washed three times with buffer solution, and adjusted to a concentration of 106 cells/mL. Subsequently, 5×105 cells were incubated with solution A (Trypsin buffer), solution B (Trypsin inhibitor and RNase buffer), and Solution C (PI stain solution) for 10 minutes respectively in the darkroom before analysis. All flow cytometry analyses were performed using BD Accuri™ C6 Plus personal flow cytometer (BD Biosciences).
4
Evaluating VLP-Cetuximab-Cy5.5 Interactions
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To check the specific interaction between VLPs, cetuximab, andCy5.5, we carried out a flow cytometry assay using two different cell lines: Cal33 (from squamous cell carcinoma, overexpressing EGFR) and THP1 (monocytes that do not express EGFR). Approximately 50,000 cells per experiment were incubated with 0.01 µg/mL of VLP–cetuximab–Cy5.5 (functionalizations A and B) for 40 min at RT. We used a BD Accuri™ C6 Plus Personal Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). We used near-red laser light as background noise and far-red laser to check for the presence of Cy5.5.
5
Ki-67 Cell Proliferation Assay in AC16 Cells
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Cell proliferation assay was performed in AC16 cells using the Ki-67 Alex Fluor 488 Conjugate antibody (11,882, cell signaling). After 7 days of treatment with HG and tirzepatide, cells were trypsinized, collected, and washed gently with PBS, resuspended in 100 µl 4% formaldehyde per 1 million cells and incubated for 15 min at room temperature. After the cell was washed with PBS, permeabilized with 90% methanol ice-cold, and incubated for 10 min on ice. Cells were washed with PBS, resuspended in 100 µl of the primary antibody with 1:50 dilution, and incubated for 1 h at room temperature and dark place. Cells were washed and resuspended in the next step with PBS for measurement that was carried out using BD Accuri C6 Plus Personal Flow Cytometer (BD biosciences) with FITC filter in Ex/Em = 480/530 nm. DData processing was performed using lowJo BD Accuri C6 Plus software for Windows.
6
Autophagy Assay in AC16 Cells
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According to the manufacturer's protocol, Autophagy Assay Kit was performed in AC16 cells using the Autophagy Assay Kit (ab139484, abcam). Briefly, cells were seeded in 6-well plates and exposed to HG and tirzepatide for 7 days. According to the manufacturer’s instructions, cells were trypsinized, collected with 100 µL of 1 × assay buffer, and centrifugated for 5 min at 400 ×g. According to the kit, protocol cells were resuspended in 250 µl of diluted green dye staining solution and incubated for 30 min at 37 C and subsequently washed two times with 1 × assay buffer and resuspended in 1X assay buffer for measurement that were carried out using BD Accuri C6 Plus Personal Flow Cytometer (BD biosciences) with FITC filter Ex/Em = 480/530 nm. Data processing was performed using FlowJo BD Accuri C6 Plus software for windows.
7
Nuclear DNA Content Determination
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Nuclear DNA content was determined with flow cytometry for the parents, hybrid and three randomly selected progeny (OCB-010, OCB-065 and OCB-180) by Heavenly Gardens (Galloway, OH, USA). Nuclei were extracted from fresh leaf tissue and nuclear DNA was stained with the CyStain® PI Absolute P reagent kit. Samples were analyzed on a BD Accuri™ C6 Plus Personal Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Laser excitation was set to 488 nm and 640 nm. Emissions were detected through 4 colors with standard optical filters: FL1 533/30 nm, FL2 585/40 nm, FL3 > 670 nm and FL4 675/25 mm. Tomato (Solanum lycopersicum) and Daylily ‘Purple Pixie Gumdrop’ (Hemerocallis) with estimated genome sizes (2C DNA content) of 2.05 Gb and 8.59 Gb, respectively, were included as internal controls for each sample.
8
Quantifying Intracellular ROS Levels
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The oxidant-sensitive fluorescent probe dihydrorhodamine (DHR; Sigma-Aldrich, St. Louis, MO, USA) was used to determine the intracellular ROS levels. Cells were treated with 50 µM DHR in culture media for 1 h, and then washed with 0.9% NaCl twice and suspended in 200 µL of 0.9% NaCl. Cells treated with 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) during 10 min before incubation with DHR were used as positive control. Fluorescence was measured in FL-1 channel (533/530 nm) in a BD Accuri™ C6 plus Personal Flow Cytometer. Data was acquired from a total of 20,000 events/samples from 2 independent assays. Quantification of intracellular ROS was expressed by relative fluorescence units (RFU) calculated in relation to sample control (unlabeled cells).
9
Quantifying Cell Proliferation by Flow Cytometry
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Cell proliferation was assayed by Ki-67 (Alexa Fluor® 488 conjugate) antibody (Cell Signaling cat#11882). According to the manufacturer’s instructions, the cells after 7 days treatment are detached and washed with PBS. The cells were then fixed with 4% formaldehyde followed by incubation for 15 min at room temperature. After washing with PBS, cells were permeabilized using 0.1% PBS-tween on ice for 15 min. According to datasheet 1 × 106 cells were incubated with the antibody for 1 h at room temperature after washing with PBS. Before measurement the cells were again washed 2 times and resuspended in PBS. The measurements were carried out using BD Accuri C6 Plus Personal Flow Cytometer (BD biosciences) and data processing was performed by FlowJo BD Accuri C6 Plus software for windows [27 (link)].
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