Cells were seeded in 24-well plates (2×105 per well) and cultured with medium containing 400 µM CoCl2 for 6 h. Apoptotic cells were measured by flow cytometry using Annexin V-APC and 7-AAD Apoptosis Detection reagent (BD Biosciences). Briefly, cell were digested, collected, washed and incubated at room temperature for 10 min with 1X binding buffer containing Annexin V-conjugated APC and 7-AAD. Annexin V-APC staining was used to detect exposure of phosphatidylserine. 7-AAD is able to penetrate the cells when cell apoptosis and cell death occurs. Annexin V-APC was used to detect early apoptotic cells, and Annexin V-APC and 7-AAD were used to detect necrotic cells or/and nonviable apoptotic cells. Cells were detected by BD Accuri C6 Flow Cytometer and analyzed by BD Accuri™ C6 Software version 1.0 (BD Biosciences).
Accuri c6 software version 1
Manufactured by BD
Sourced in Italy, United States
The BD ACCURITM C6 software version 1.0 is a data analysis tool designed to work with BD flow cytometry instruments. It provides users with the ability to analyze and interpret data generated from flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 flow cytometer, by BD (4 mentions)
Accuri c6, by BD (4 mentions)
Annexin 5 apc and 7 aad apoptosis detection reagent, by BD (1 mentions)
Syto 24, by Thermo Fisher Scientific (1 mentions)
Mitochondrial membrane potential detection jc 1 kit, by BD (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Fitc conjugated annexin 5 and pi, by BD (1 mentions)
10 protocols using accuri c6 software version 1
1
Quantifying CoCl2-Induced Apoptosis by Flow Cytometry
2
Flow Cytometric Analysis of Cell Viability
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Flow cytometric analysis was conducted using a BD Accurri™ C6 sampler Plus (BD; Franklin Lakes, NJ, USA) on NWS samples. Samples were diluted in phosphate-buffered saline (PBS) to obtain a final concentration between 105 and 106 cells/mL. Samples were stained separately with two pairs of fluorescent markers of propidium Iodide (PI; Life Technologies, Carlsbad, CA, United States)/Syto™ 24 (Life Technologies, Carlsbad, CA, United States) and 6-Carboxyfluorescein diacetate (CFDA; Merck, USA)/PI. Treated samples were incubated at 37 °C while shaking and protected from light for 15 min. All Samples were analyzed by performing Protocol A and Protocol B according to ISO 19344:2015 [16 ] specifications. A 50 μL of NWS diluted samples was taken for instrument reading. The thresholds for side scattering (SSC) and forward scattering (FSC) were 1000 and 3,000, respectively. Moreover, an excitation laser at 488 nm and an emission filter at 533/30 (FL1) and 675/25 (FL3) were used to visualize the stained events. Data was collected and analyzed using BD Accuri™ C6 software version 1.0 (BD Biosciences, USA).
3
Intracellular ROS Measurement Assay
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To perform the assay, the cells, seeded in 6-well plates (1 × 106 cells/well), were treated with the tested substances (β-caryophyllene, 50 µM; doxorubicin, 20 µM) for the required time exposure, then the ROS levels were measured in the pellets by the 2,7-dichlorofluorescein diacetate assay (DCFH-DA), as previously reported [61 ]. Intracellular ROS proportionally reduced DCFH-DA to the fluorophore DCF, whose fluorescence was measured at an excitation wavelength of 485 nm and emission wavelength of 528 nm by a BD Accuri™ C6 flow cytometer (BD Biosciences, Milan, Italy). In each experiment, a vehicle control, corresponding to a basal ROS level, were included too. For all the treatments, the mean DCF fluorescence of 50,000 cells was determined by a BD AccuriTM C6 Software version 1.0.264.21 (BD Biosciences, Milan, Italy).
4
Mitochondrial Membrane Potential Measurement
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Changes of ΔΨm in cells were performed using the Mitochondrial Membrane Potential Detection JC-1 Kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instruction. In short, following incubation with QDs, UAs, and QD−UA hybrids at IC80 value for 24, 72, and 144 h, cancer cells were collected and centrifuged at 400 g for 5 min at 23 °C. Next, the cells were stained with 500 µL working solution (contained 5 μL JC-1 dye and 495 μL 1× assay buffer) and incubated at 37 °C in a CO2 incubator for 15 min. Afterward, the cells were washed twice with 1× assay buffer (2 mL and 1 mL, respectively), resuspended with 450 μL 1× assay buffer, and analyzed by flow cytometry (Accuri™ C6; Becton Dickinson, San Jose, CA, USA). The data were analyzed using BD AccuriTM C6 Software Version 1.0.264.21 (San Jose, CA, USA). Each experiment was repeated at least three times.
5
Yeast Cell Physiological Assessment via Flow Cytometry
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Flow cytometry (FCM) was used to monitor the physiological state of yeast cells immediately after PEF and after a 6 h recovery, to assess if cells were able to restore the damage caused by such treatments. Cell suspensions collected immediately after treatments and during recovery were analyzed with a flow cytometer Accuri C6 (BD Biosciences, Milan, Italy), using setting parameters, emission filters and thresholds according to Arioli et al. (2019) (link). The cells were stained with SYBR-Green I (1X), propidium iodide (PI) 7.5 μM, and DiBAC4(3) [Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol] 3.0 μM as reported by Tabanelli et al. (2019) (link), and data obtained were analyzed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy).
6
Cell Viability Assay with Flow Cytometry
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To test cell viability, the samples were collected after 24, 48, and 72 h of incubation and analyzed in triplicate with a flow cytometer Accuri C6 (BD Biosciences, Milan, Italy), following the protocol reported by Arioli et al. (41 (link)). Before analyses, samples were diluted (if needed) in filtered PBS, and the cells were stained with SYBR-Green I (1X) and propidium iodide (7.5 μM) at 37°C for 15 min. This dual staining allowed to differentiate three sub-populations corresponding to different physiological states: live, injured, and dead cells. The data obtained were analyzed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy).
7
Flow Cytometry Monitoring of Lactobacillus sakei
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Flow cytometry (FCM) was used to monitor the physiological state of L. sakei Chr82 cells in each sample. Cell suspensions were analyzed with the flow cytometer Accuri C6 (BD Biosciences, Milan, Italy), using setting parameters, emission filters and thresholds according to Arioli et al. [15 (link)].
Before the analysis, where necessary, the samples were diluted in the corresponding DM up to a concentration of 7 log CFU/mL, the optimal cell density for a correct sample staining by fluorochromes.
The cells were stained with SYBR-Green I (1X), propidium iodide (PI) 7.5 μM and DiBAC4 (3) (Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol) 3.0 μM as reported by Tabanelli et al. [16 (link)]. The data obtained were analyzed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy). Before analysis, each aliquot was kept at 37 °C for 15 min in order to let the dye react with the cells.
Before the analysis, where necessary, the samples were diluted in the corresponding DM up to a concentration of 7 log CFU/mL, the optimal cell density for a correct sample staining by fluorochromes.
The cells were stained with SYBR-Green I (1X), propidium iodide (PI) 7.5 μM and DiBAC4 (3) (Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol) 3.0 μM as reported by Tabanelli et al. [16 (link)]. The data obtained were analyzed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy). Before analysis, each aliquot was kept at 37 °C for 15 min in order to let the dye react with the cells.
8
Flow cytometric analysis of plant derivatives
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Cell suspensions collected after 24, 48, and 72 h of incubation in the presence of the different plant derivatives were analysed with a flow cytometer Accuri C6 (BD Biosciences, Milan, Italy), using setting parameters, emission filters, and thresholds according to Arioli et al.32 (link). Before analyses, samples were diluted (if needed) in filtered PBS and the cells were stained with SYBR-Green I (1×) and propidium iodide (PI) 7.5 µM at 37 °C for 15 min, in order to let the dye react with the cell. This dual staining allowed to distinguish three sub-populations corresponding to different physiological states: live, injured, and dead cells. The data obtained were analysed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy).
9
B16F10 Apoptosis Assay Protocol
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B16F10 cells (1×105) were seeded into 12-well plates and cultured overnight in 5% CO2 at 37°C. Cells were treated with ICRP (1 U/ml), OXP (800 µM) or a combination of ICRP (1 U/ml) + OXP (800 µM) for 24, 48 and 72 h. Following treatment, cells were collected and washed with phosphate-buffered saline (PBS) and resuspended in 100 µl of 1X binding buffer (0.1 M Hepes pH 7.4, 1.4 M NaCl and 25 mM CaCl2; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with APC-conjugated Annexin V (5 µl/sample) and propidium iodide (1 µl/sample), incubated on ice and kept in the dark for 15 min. Flow cytometry analysis was performed using an Accuri C6 cytometer; BD Accuri C6 Software version 1.0.264.21 was used for data analysis (both BD Biosciences, San Jose, CA, USA).
10
Annexin V-FITC/PI Apoptosis Assay
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H1299 and H460 cells (1×105 per well in a 6-well plate) were collected following incubation with CPT and/or 3-MA for 24 h. The cells were washed with PBS and were then resuspended in Annexin V binding buffer [140 mM NaCl, 10 mM HEPES-NaOH (pH 7.4) and 2.5 mM CaCl2]. Following 300 × g centrifugation for 5 min, the cells were incubated with Annexin V binding buffer containing 1.25 ml fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI (BD Pharmingen; BD Biosciences, San Jose, CA, USA) at room temperature for 15 min in the dark. Data acquisition and analysis were performed using the BD Accuri™ C6 flow cytometer with BD Accuri™ C6 software version 1.0.264.21 (both from BD Biosciences).
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