Membrane potential was assessed using a flow cytometry assay based on the BacLight bacterial membrane potential kit (Life Technologies). Cells from overnight cultures were inoculated in 10 ml TSB in 100-ml Erlenmeyer flasks and grown to an optical density at 600 nm (OD600) of 0.2. Fifteen microliters of culture was transferred to 1 ml filtered PBS. To each cell solution, 10 μl of the fluorescent membrane potential indicator dye DiOC2 (3 (link)) was added and cells were stained for 5 min at room temperature. Data were recorded on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Settings on the flow cytometer were as follows: 50,000 recorded events at a forward scatter (FSC) threshold of 15,000 and medium flow rate. Gating of the stained cell population and analysis of flow cytometry data were performed in CFlow (BD Accuri). As an indicator of membrane potential, the ratio of red to green fluorescence intensity was calculated. The assay was verified with the NTML mutant containing a transposon insertion in menD (NE1345), which displays depolarization of the membrane (45 (link)).
Biosciences accuri c6 flow cytometer
Manufactured by BD
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The BD Biosciences Accuri C6 flow cytometer is a compact and user-friendly instrument designed for analyzing particle and cell populations. It employs flow cytometry technology to measure and analyze various characteristics of cells or particles suspended in a fluid stream.
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5 protocols using biosciences accuri c6 flow cytometer
1
Membrane Potential Assessment by Flow Cytometry
2
Membrane Potential Assay via Flow Cytometry
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Assessment of variations in membrane potential was estimated using a flow cytometry assay based on the BacLight Bacterial Membrane Potential Kit (LifeTechnologies). Cells from ON cultures were inoculated in 30 ml TSB in 300 ml Erlenmeyer flasks and grown to an OD600 of 0.2. Fifteen microliter culture was transferred to 1 ml filtered phosphate-buffered saline (PBS). To each cell solution 10 μl of fluorescent membrane potential indicator dye, DiOC2(3), was added and cells were stained for 30 min at room temperature. Data was recorded on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Settings on the flow cytometer were as follows: 50000 recorded events at a FSC threshold of 15000 and medium flow rate. Gating of stained cell population and analysis of flow cytometry data were performed in CFlow® (BD Accuri). As an indicator of membrane potential the ratio of red to green fluorescence intensity was calculated. The assay was verified using the two SCV strains with transposon insertions in hemB (NE1845) and menD (NE1345).
3
Detecting Mitochondrial ROS Production
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Mitochondrial-derived ROS (mtROS) production was detected by staining the cells with MitoSOX™ Red reagent (Invitrogen, Waltham, MA, USA). About 5 × 104 fibroblasts or 1 × 105 SH-SY5Ycells were plated in duplicates in a 24-well plate, and after 24 h of seeding, incubated in the dark at 37 °C for 20 min with 10 μM and 5 μM of the probe, respectively. Negative controls were also included. After staining, the cells were washed, and fluorescence was detected in each sample using a flow cytometry-based method. Cells were analyzed within 10–20 min after completion of MitoSOX staining, avoiding possible nuclear accumulation. In parallel, the cell lines were incubated with antimycin A (20 μM) during the last 15 min of the MitoSOX staining to increase mtROS production. A minimum of 5000 gated events was collected on a BD Biosciences Accuri™ C6 flow cytometer (Becton Dickinson, San Jose, CA, USA). All data were analyzed with BD Accuri™ C6 Plus Analysis software (Becton Dickinson). Measurements were normalized by subtracting the blanks.
4
Bacterial Membrane Potential Assay
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Differences in membrane potential between the strains were estimated using a flow cytometry assay based on the BacLight Bacterial Membrane Potential Kit according to the manufacturer (Life Technologies®, cat. no. B34950). Briefly, bacterial strains were grown overnight shaking at 37°C in TSB. Fifty μL of cells from each overnight culture was inoculated into 10 mL of TSB in 100 mL Erlenmeyer flasks and grown with shaking at 37°C to an OD600 of 0.2. Then 15 μL of culture was transferred to 1 mL of PBS and 10 μL of fluorescent membrane potential indicator dye, DiOC2(3) was added. Cells were stained for 30 min at room temperature followed by analysis on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Fifty-thousand events were recorded at a forward scatter threshold of 15 000 and medium flow rate. Gating of stained cell population and analysis of flow cytometry data were performed in CFlow® (Becton, Dickinson and Company). As an indicator of membrane potential, the ratio of red to green fluorescence intensity was calculated. The assay was verified using the protonophore CCCP and two strains derived from the Nebraska Transposon Mutant Library25 (link) that display reduced membrane potential (ΔmenD, NE1345 and ΔhemB, NE184532 (link)).
5
Doxorubicin Cytotoxicity Assay in Cell Lines
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Cells (3x 10 4 /flasks) were cultured in T25 flasks over 24hrs, and then exposed to a DOX dose corresponding to the inhibitory concentration, IC 50 , adjusted to the cell number [29] , determined by cytotoxicity assays for each time point (from 2hrs to 72hrs) and each cell line.
After each incubation period, cells were trypsinised and centrifuged in 5 mL fresh medium at 4°C and 1100 rpm for 5 min and they were then re-suspended in1mL Ice Cold Dulbecco's Phosphate-Buffered Saline (DPBS) buffer and centrifuged at 4°C and 2500 rpm for 5 min.
Cells were re-suspended in 750 µL ice cold DPBS buffer and transferred to Eppendorf tubes to which 250 µL of fixation buffer were added. After 30 min incubation at 4ºC, the fixed cells were washed twice in perm/wash buffer, centrifuged (2500 Rpm for 5mn at 4ºC) and then gently re-suspended in 50 µL perm/wash buffer, after which 20 µL of the antibody were added and the cells were incubated for 60 min in the dark at 4ºC. The cells were then washed twice in perm/wash buffer, centrifuged (2500 Rpm for 5mn at 4ºC) to remove unbound antibody and finally re-suspended in 1mL stain buffer. 10,000 cells were analysed by Flow Cytometry using a BD Biosciences Accuri C6 Flow Cytometer (Becton Dickinson, Oxford, UK). The Accuri Flow Cytometry software was used for the analysis of Flow Cytometry samples and data processing.
After each incubation period, cells were trypsinised and centrifuged in 5 mL fresh medium at 4°C and 1100 rpm for 5 min and they were then re-suspended in1mL Ice Cold Dulbecco's Phosphate-Buffered Saline (DPBS) buffer and centrifuged at 4°C and 2500 rpm for 5 min.
Cells were re-suspended in 750 µL ice cold DPBS buffer and transferred to Eppendorf tubes to which 250 µL of fixation buffer were added. After 30 min incubation at 4ºC, the fixed cells were washed twice in perm/wash buffer, centrifuged (2500 Rpm for 5mn at 4ºC) and then gently re-suspended in 50 µL perm/wash buffer, after which 20 µL of the antibody were added and the cells were incubated for 60 min in the dark at 4ºC. The cells were then washed twice in perm/wash buffer, centrifuged (2500 Rpm for 5mn at 4ºC) to remove unbound antibody and finally re-suspended in 1mL stain buffer. 10,000 cells were analysed by Flow Cytometry using a BD Biosciences Accuri C6 Flow Cytometer (Becton Dickinson, Oxford, UK). The Accuri Flow Cytometry software was used for the analysis of Flow Cytometry samples and data processing.
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