The liver samples of rats were harvested before blood collection. Briefly, liver tissues of equal sizes were taken from the same part of the left lobe of the rat livers and rapidly placed in a 1640 medium containing 5% fetal bovine serum (FBS). The liver tissues were moved from the medium onto a 300-mesh filter and ground gently to let the cells pass through the filter. Afterward, the cells were collected and washed with phosphate buffer saline (PBS). 2×106 cells were placed in the flow tube and washed with PBS. Then, 1 μL Annexin V YO-PRO-1 dye and 1 μL PI dye from Vybrant® Apoptosis Assay Kit #4 (Invitrogen, Carlsbad, CA, USA) were added to the flow tubes. The cells were static-cultured at 4 °C in the dark for 20 min and resuspended in PBS. Finally, the hepatocyte apoptosis rate was detected by flow cytometry-BD FACS Canto II Plus (BD Biosciences, San Jose, CA, USA).
Facs canto 2 plus
Manufactured by BD
Sourced in United States
The FACS Canto II plus is a flow cytometry instrument designed for cell analysis and sorting. It is a versatile and reliable tool for researchers and clinicians in various fields, such as immunology, hematology, and cell biology. The FACS Canto II plus is capable of detecting and analyzing multiple parameters of individual cells or particles within a sample.
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Lab products found in correlation
Annexin 5 fitc, by BD (2 mentions)
Vybrant apoptosis assay kit 4, by Thermo Fisher Scientific (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Mito tracker red cmxros, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Cell cycle kit, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Binding buffer, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
7 protocols using facs canto 2 plus
1
Rat Liver Hepatocyte Apoptosis Assay
2
Quantifying CAR-T Cell Cytotoxicity against Tumor Cells
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CAR-T cells were co-cultured with or without NALM-6-eGFP (2:1) in a 24-well plate. After 24 h, cells were collected and tumor cells were detected by surface markers using flow cytometry (BD FacsCanto II Plus) per the manufacturer’s instructions.
CAR-T cells were co-cultured with or without NALM-6-eGFP (2:1) in a 96-well plate. After 24 h, the luciferases activities were monitored using the IVIS imaging system (IVIS, Xenogen, Alameda, CA, USA) after adding 20 μL D-luciferin potassium (1.515 mg/mL) (Thermo Fisher Scientific). Cell viability = fluorescence value of live cells/control × 100%
CAR-T cells were co-cultured with or without NALM-6-eGFP (2:1) in a 96-well plate. After 24 h, the luciferases activities were monitored using the IVIS imaging system (IVIS, Xenogen, Alameda, CA, USA) after adding 20 μL D-luciferin potassium (1.515 mg/mL) (Thermo Fisher Scientific). Cell viability = fluorescence value of live cells/control × 100%
3
Potassium, Mitochondria, and ROS in HK-2 Cells
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Potassium level: HK‐2 cells were cultured in 6‐well plates, treated with heme, and inhibited for 4 h. Then, the supernatant was collected to measure potassium levels using a kit (Abacam, #ab252904, UK). Mitochondria damage: Briefly, the HK‐2 cells in different groups were incubated at 37°C for 15 min with 200 nmol/L Mito‐Tracker Red CMXRos (#C1049, Beyotime, China) and were collected and detected by flow cytometry (BD, FACS Canto II plus, United States). Intracellular ROS levels: According to the Reactive Oxygen Species Assay Kit (#S0033S, Beyotime, China), the HK‐2 cells in different groups were incubated at 37°C for 15 min with 10 μmol/L DCFH‐DA (included in #S0033S, Beyotime, China), and flow cytometry was used to detect intracellular ROS levels. For the viability assay, HK‐2 cells in different groups were cultured in 96‐well plates, treated with heme, and inhibited for 4 h. Then, cell viability was assessed using the kit (Boster Biological Technology Co. Ltd., Wuhan, China). N‐acetyl‐l ‐cysteine (NAC) (#A9165, Sigma‐Aldrich), an inhibitor of ROS, was used to detect the changes in ROS and cell viability after different stimulations.
4
Cell Cycle Analysis of HCT-116 and HEK293T Cells with HRS Treatment
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The HCT-116 cells and HEK293T cells with a density of 3 × 105 cells/well were exposed to HRS (0, 50, 90, or 130 μM) for 24 h at 37°C. Then, cells were treated with trypsin, gathered, and fixed with 5 mL 80% ethanol at 4°C for 18 h, followed by washing with PBS. Propidium iodide (PI) was added and cells incubated for 30 min in the dark. Cell cycle distribution was assessed by flow cytometry (FACS Canto II plus, BD BioSciences, USA) with a cell cycle kit (BD, NJ, USA).
5
Apoptosis Assay of HCT-116 Cells
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After treatment with a series of HRS solutions (0, 50, 90, or 130 μM) for 24 h, the HCT-116 cells were harvested. Washed cells were resuspended with 400 μL of 1 × Annexin V binding buffer at a final concentration of 3 × 105 cells/mL. Then, 5 μL Annexin V-FITC (BD, New Jersey, USA) was added and incubated in the dark for 15 min at 2–8°C. Thereafter, 5 μL PI (BD, New Jersey, USA) was added to the mixture and incubated for 10 min. Cell apoptosis was examined using flow cytometry (FACS Canto II plus, BD BioSciences, USA).
6
miR-148a-3p Modulates Cell Apoptosis
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To analysis the effects of miR-148a-3p in cell apoptosis by Annexin V-FITC/PI staining assay (BD, New Jersey, USA). After transfection, cells from the different treatment groups (three independent replicates per treatment) were washed three times with PBS buffer (pH=7.4), collected by trypsinization, washed again with PBS, and then resuspended in 1ml 1×binding buffer (BD, New Jersey, USA). Afterward, we incubated the cells for 15 min in the dark at room temperature in the presence of Annexin V-FITC (5μl) and propidium iodide (PI) (5μl, BD, New Jersey, USA). Afterward, cells were analyzed using ow cytometry (FACS Canto II plus, BD BioSciences, USA).
7
miR-148a-3p Modulates Cell Cycle
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To analysis the effect of miR-148a-3p in different periods of the cell cycle, we analyzed the cell cycle with miR-148a-3p mimics, mimics NC, inhibitor, inhibitor NC groups using the cell cycle testing kit (BD, New Jersey, USA). The cells cultivated in six-well plates were collected and then centrifuged at 800g/min for 5min. The supernatant was discarded, and the cells were washed with PBS and xed with 75% alcohol for 24 hours. The xed cells were centrifuged at 1000g/min for 10min, washed with PBS to remove alcohol, stained with PI/RNase for 15 minutes in the dark at room temperature, and detected for ow cytometry (FACS Canto II plus, BD BioSciences, USA).
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