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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein
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NOTE: This protocol was optimized for 70%–80% confluent MDA-MB-231 cells. For other cell lines, the number of cells and multiplicity of infection (MOI) should be reoptimized.

1. Preparation of cells (day 1)

  1. Maintain MDA-MB-231 cell line in 75 cm2 flasks with 10 mL of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 humidified atmosphere.
    NOTE: DMEM supplemented with 10% FBS, 37 °C, and a 5% CO2 humidified atmosphere are used for all attachment and treatment incubations throughout the entire protocol.
  2. Prepare the MDA-MB-231 cells for experiment.
    1. Aspirate the medium within the flask, detach the cells with 2 mL of 0.25% trypsin-EDTA solution for 2 min, and inactivate the trypsin activity with 6 mL of complete medium (DMEM with 10% FBS). Centrifuge the cells at 150 x g for 5 min. Aspirate the supernatant and suspend the cells in 5 mL of complete medium.
    2. Mix an equal volume cell suspension and 0.4% trypan blue. Take 10 µL of this mixture and count the cells with the automated cell counter.
      NOTE: A Coulter counter or a hemocytometer can also be used for cell counting.
    3. Seed the cells into a 6 well plate for flow cytometry analyses and seed 150,000 cells in 1 mL of medium per well. Wait 16 h for cell attachment.
    4. Seed the cells into a 4 well chamber slide for fluorescent imaging and seed 25,000 cells in 0.5 mL of medium per well. Wait 16 h for cell attachment.
      NOTE: Seed control wells in addition to treatment wells. Use one of the control wells to determine cell number (optional: if the attachment period for the cells is shorter than the doubling time, cell number can be assumed to be the same as the seeding density) and the other for a noninfected control (0 MOI).

2. Adenoviral roGFP transduction (day 2 and 3)

CAUTION: Adenoviruses can cause diseases. While transducing the cells, use filtered tips and decontaminate tips, Pasteur pipettes, and microcentrifuge tubes with 10% bleach.

NOTE: This protocol was demonstrated with cytosol-specific roGFP, but other cellular compartments (e.g., mitochondria or mitochondrial intermembrane space) can be targeted with this same protocol.

  1. Generate a dose-response curve for the MOI to obtain the highest transduction efficiency by calculating the volume of adenovirus (mL) required for each MOI value for MDA-MB-231 cell line (Table 1):
    Equation 1
    NOTE: Functional titer of each batch of adenoviral stock, which is expressed as plaque forming unit (PFU) per mL, is provided by the company. The optimum MOI for transduction differs between cell types. For most mammalian cells, the optimum MOI range is between 10 and 300. According to the cellular response, MOI values should be recalculated (e.g., MOI range should be reduced if cells have cytotoxic response, or range should be increased if cells have low transduction efficiency).
  2. Make 1:100 dilution of 6 x 1010 PFU/mL adenoviral roGFP solution with cell culture medium (DMEM with 10% FBS) for reliable pipetting.
  3. Pipette and add 0.0125 mL (12.5 µL), 0.025 mL (25 µL), 0.05 mL (50 µL) of adenoviral roGFP dilution into each well of the 6 well plate in order to transduce the 150,000 cells with 50, 100, and 200 MOI respectively for flow cytometry analysis (Table 1).
  4. Pipette and add 0.0042 mL (4.2 µL) of adenoviral roGFP dilution in the 4-chamber slide wells to transduce 25,000 cells with 100 MOI for fluorescence imaging (Table 1).
    NOTE: A minimal amount of medium should be used in the wells to ensure the highest interaction between the adenoviral roGFP construct and cells. The serum content of the culture medium may need to be decreased for different cell lines because high levels of serum can negatively affect transduction efficiency in some cell types.
  5. Incubate cells for 16–24 h under the cell maintenance conditions. The next day (day 3), change the medium to cell culture medium (DMEM with 10% FBS) to allow cell recovery for an additional 24 h. Visualize cells under a microscope to assess their morphology; cells can express roGFP even if they have morphological changes.
    NOTE: On day 3, cells should start to express roGFP; therefore, transduction efficiency can be monitored using fluorescence microscopy (filters with ex. 488/em. 525). To obtain consistent assay results, be aware of and document the morphological changes under the phase contrast microscope and observe morphology while evaluating transduction efficiency.
  6. Construct a dose response curve using the 50, 100 and 200 MOI samples prepared in step 2.3 and their transduction efficiency results obtained from flow cytometry analysis (steps 3.1 and 4.1). Assess optimal transduction efficiency with documentation of morphological changes (step 2.5) and the dose-response curve of MOI.
    NOTE: Although more than 98% of the cell population at 100 MOI and 200 MOI express roGFP (see representative results), 200 MOI group showed substantial changes in cell morphology of MDA-MB-231 cells. Consequently, the most efficacious MOI for MDA-MB-231 cells was determined to be 100 MOI.
  7. After optimal MOI (here, 100 MOI) was chosen for MDA-MB-231 cell line, conduct experiment with test materials (10 µM H2O2 and its vehicle 0.1% deionized water).
    1. Prepare and seed the cells according to section 1. Using the adenoviral transduction volume for 100 MOI calculated in step 2.1, repeat steps 2.2−2.4 for 100 MOI adenoviral transduction of cells. Then incubate the plate and chamber slides according to step 2.5.

3. Acquisition of CyS/CySS balance

  1. Flow cytometry (day 4)
    1. On day 4, incubate cells from step 2.7.1 with 10 µM H2O2 for 1 h.
      NOTE: 10 µM H2O2 was used as the test substance and 0.1% deionized water was used as vehicle treatment in this protocol. Other oxidizing agents can be used as positive controls here.
    2. Aspirate media from the 6 well plate, replace with 750 µL of 0.25% trypsin-EDTA solution and wait for 2 min for cells to detach. Inactivate trypsin with 2 mL of complete medium (DMEM with 10% FBS) and collect the volume into 15 mL conical tubes.
    3. Centrifuge the tubes at 150 x g for 5 min at 4ºC. Discard supernatant and suspend the cells in 500 µL of phosphate-buffered saline (PBS).
    4. Repeat step 3.1.3
    5. Filter the cell suspensions into flow cytometry-compatible tubes using 40 µm mesh. Keep the tubes on ice and away from the light and follow step 4.1 for data analysis.
  2. Microscopic imaging (day 4)
    1. On day 4, treat cells with 10 µM H2O2, acquire images immediately (time point 0) and 1 h after treatment and follow step 4.2 for data analysis.

4. Data analysis

  1. Flow cytometry quantification
    1. Set flow cytometry method for 3 different analyses via sample aquisition software (see Table of Materials): forward scatter (FCS) on x-axis and side scatter (SSC) on y-axis to assess cell size and complexity of cells (SSC can be used for rough identification of dead and live cells); ex. 488 nm/em. 525 nm (fluorescein isothiocyanate [FITC]) bandpass filter on x-axis and SSC on y-axis to assess CyS-roGFP; ex. 405 nm/em. 525 nm (Brilliant Violet 510 [BV510]) bandpass filter on x-axis and SSC on y-axis to assess CySS-roGFP.
    2. Acquire 0 MOI control and visualize cells with sample acquisition software. Repeat this step for remaining samples (50, 100, 200 MOI groups and later on 10 µM H2O2 treated cells and vehicle treated cells). Save the files for data analysis.
    3. Open data analysis software (see Table of Materials) and open 0 MOI sample file. Assess cell population of interest (Gate 1). Set up the following gatings to minimize background fluorescence for ex. 488 nm/em. 525 nm (Gate 2) and ex. 405 nm/em. 525 nm (Gate 3) bandpass filters with the noninfected (0 MOI) control cells.
    4. Open 50, 100, and 200 MOI sample files within data analysis software to assess the dose-response curve. Analyze mean fluorescence intensities with Gates 2 and 3 for each sample. Repeat this step for test samples (10 µM H2O2 treated cells and vehicle treated cells).
    5. Calculate the mean fluorescent intensity ratio between oxidized versus reduced forms of roGFP with the following equation.

Equation 2

  1. Image assessment
    1. Use a microscope that contains fluorescence filters for CyS-roGFP and CySS-roGFP (ex. 488 nm/em. 525 nm and ex. 405 nm/em. 525 nm filters, respectively).
    2. In each well of the chamber slide, pick 4 random areas to acquire images, using the 4x objective to visualize larger areas.
      NOTE: 20x objective can also be used for image displays.
    3. Open the image with ImageJ software11. Apply Analyze | Measure commands for each image and use the equation in step 4.1.5 to quantify the data.
      NOTE: Quantification of the images is ratiometric; therefore, the protocol does not include subtraction of background. However, to be able to compare images, brightness, contrast, and saturation must be the same for each image. Statistical significance was assessed with one-way analysis of variance (ANOVA) and Tukey’s post hoc test.

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Learning Objectives

The redox state of CyS/CySS is easily assayed with transduced roGFPs. The fluorescent probe quantifies the ratio between the reduced and oxidized forms (excitation wavelengths 488 nm and 405 nm, respectively). Fluorescence data can be obtained by both flow cytometry and microscopy.

A large number of cells can consistently and conveniently be acquired using flow cytometry. The analysis consists of 3 main steps: 1) select the cell population of interest with the FSC area filter (Figure 1A); 2) gate the roGFP-expressing cells with ex. 488/em. 525 nm with a selective bandpass filter (Figure 1B); and 3) gate the oxidized roGFP-containing cells from the roGFP-expressing cells with ex. 405 nm/em. 525 nm bandpass filter (Figure 1C).

Each new cell line should be evaluated for the optimum adenoviral transduction efficiency of roGFPs. Transduction efficiency should be assessed with morphological evaluation of cells and roGFP expression analyses with flow cytometry and/or fluorescent microscopy. This protocol uses flow cytometry to determine the dose-response curve for roGFP analyses and to select the most efficient MOI input (Figure 2A−H). According to the MOI dose-response curve (Figure 2I), 200 MOI gave the highest roGFP expression, but cell morphology was affected, suggesting cytotoxicity. Therefore, the optimum transduction efficiency was determined to be with 100 MOI.

To evaluate the effectiveness of the method, H2O2 was used as a positive control for oxidation. One hundred MOI was used for optimum transduction. After the recovery period, cells were treated with 10 µM H2O2 for 1 h to obtain the fluorescence ratio via flow cytometry. Oxidized (ex. 405 nm/em. 525nm) and reduced (ex. 488 nm/em. 525 nm) roGFP mean fluorescence intensities were obtained from flow cytometry analyses for vehicle (Figure 3A, B) and 10 µM H2O2 (Figure 3C, D) treatments. The overlaid histograms represent the shift in the cell numbers of 10 µM H2O2 and vehicle treated groups for reduced (Figure 3E) and oxidized (Figure 3F) roGFP. The ratio between oxidized and reduced roGFP shows that 10 µM H2O2 caused a 3-fold increase in oxidation of roGFP compared to vehicle treatment (Figure 3G).

Fluorescent imaging of cells was also performed with 10 µM H2O2 under the microscope for 1 h. Images were taken under the 4x objective, and representative images were taken under the 20x objective (Figure 4A). Fluorescent intensities were evaluated with ImageJ software, and ratios were calculated. A steady state increase in H2O2-induced oxidation was detected (Figure 4B); incubation with H2O2 for 1 h increased the oxidization of roGFP cysteines, which exhibited significant change between vehicle controls.

Figure 1
Figure 1: Gating setup for fluorescent intensities of CyS-containing (reduced) roGFP and CySS-containing (oxidized) roGFP residues with non-transduced MDA-MB-231 cells. (A) The cell population of interest was selected as Gate 1 with SSC and FSC area filters. (B) roGFP-expressing cells were selected according to non-expressing cells as Gate 2 with the ex. 488/em. 525 nm bandpass filter. (C) Oxidized (cystine) roGFP-containing cells were gated with the ex. 405 nm/em. 525 nm bandpass filter from the roGFP-expressing population. Please click here to view a larger version of this figure.

Figure 2
Figure 2: MOI dose-response curve assessment with flow cytometry analyses for MDA-MB-231 cell line. (A,B) Noninfected cells and (C,D) 50 MOI, (E,F) 100 MOI, and (G,H) 200 MOI roGFP-expressing cell populations acquired for gating setup, respectively. (I) Transduced cells were evaluated and plotted as a percentage according to the cell population of interest. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Flow cytometry assessment of CyS/CySS balance in roGFP-transduced MDA-MB-231 cell line. Vehicle-treated cells were evaluated as (A) % roGFP-expressing cells, and (B) % oxidized roGFP-expressing cells and H2O2 treatment were assessed with the same parameters in panels (C) and (D) respectively. Cell count histograms of vehicle and H2O2 treatment were overlaid for (E) reduced roGFP ex. 488/em. 525 bandpass filter and (F) oxidized roGFP ex. 405/em. 525 bandpass filter. (G) Mean fluorescence intensity ratios between oxidized/reduced forms were plotted into a bar graph. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Fluorescent imaging of roGFP-transduced MDA-MB-231. (A) Representative images after 1 h treatment with vehicle or H2O2. (B) Ratios between oxidized/reduced forms were evaluated in 4 randomly chosen areas, and bars represent mean ± standard deviation. Statistical significance between groups indicated as *(p < 0.05), **(p < 0.01), or ***(p < 0.005). Please click here to view a larger version of this figure.

Analysis type Cell number per well Adenoviral roGFP PFU/mL 1:100 dilution of adenoviral roGFP PFU/mL MOI Transduction volume (mL)
Flow cytometry 150,000 6 x 1010 6 x 108 0 0
50 0.0125
100 0.025
200 0.05
Fluorescence microscopy 25,000 6 x 1010 6 x 108 100 0.0042

Table 1: Calculation of MOI values.

List of Materials

Print Materials List
0.25% Trypsin-EDTA Gibco by Life Sciences 25200-056 Cell culture
4-well chamber slide Thermo Scientific 154526 Cell seeding material for fluorescent imaging
5 ml tubes with cell strainer cap Falcon 352235 Single cell suspension tube for flow cytometry analysis
6-well plate Corning 353046 Cell seeding material for flow cytometry analysis
15 ml conical tubes MidSci C15B Cell culture
75 cm2 ventilated cap tissue culture flasks Corning 4306414 Cell culture
Adenoviral cytosol specific roGFP ViraQuest VQAd roGFP roGFP construct kindly provided by Dr. Schumaker
Class II, Type A2 Safety Hood Cabinet Thermo Scientific 1300 Series A2 Cell culture
Countess automated cell counter Invitrogen C10227 Cell counting
Countess cell counter chamber slides Invitrogen C10283 Cell counting
DMEM Gibco by Life Sciences 11995-065 Cell culture
FBS Atlanta Biologicals S11150 Cell culture
Filtered pipette tips, sterile, 20 µl Fisherbrand 02-717-161 Cell culture
Filtered pipette tips, sterile, 1000 µl Fisherbrand 02-717-166 Cell culture
Flow Cytometer BD Biosciences LSRFortessa Instrument equipped with FITC and BV510 bandpass filters for flow cytometry analyses
Fluorescent Microscope Advanced Microscopy Group (AMG) Evos FL Fluorescent imaging
Hydrogen Peroxide 30% Fisher Scientific H325-100 Positive control
Light Cube, Custom Life Sciences CUB0037 Fluorescent imaging of roGFP expressing cells (ex 405 nm)
Light Cube, GFP Thermo Scientific AMEP4651 Fluorescent imaging of roGFP expressing cells (ex 488 nm)
MDA-MB-231 American Tissue Culture Collection HTB-26 Human epithelial breast cancer cell line
Microcentrifuge tubes, 2 ml Grenier Bio-One 623201 Cell culture
PBS Gibco by Life Sciences 10010-023 Cell culture
Pipet controller Drummond Hood Mate Model 360 Cell culture
Serologycal pipet, 1 ml Fisherbrand 13-678-11B Cell culture
Serologycal pipet, 5 ml Fisherbrand 13-678-11D Cell culture
Serologycal pipet, 10 ml Fisherbrand 13-678-11E Cell culture
Tissue Culture Incubator Thermo Scientific HERACell 150i CO2 incubator for cell culture
Trypan blue stain 0.4% Invitrogen T10282 Cell counting

Preparação do Laboratório

Print Setup Guide

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

Procedimento

Print Procedure Steps

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

Tags

Keywords: Subcellular CompartmentRedox-sensitive GFPOxidative StressRedox StateRatiometricNon-disruptiveTransductionFlow CytometryHydrogen Peroxide