Cellular Redox Profiling Using High-content Microscopy

Summary

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology – jointly referred to as mitochondrial morphofunction – in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) and tetramethylrhodamine methylester (TMRM).

Abstract

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H₂DCFDA (ROS) and TMRM (ΔΨ_m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

Introduction

The concentration of intracellular ROS is meticulously regulated through a dynamic interplay between ROS producing and ROS defusing systems. Imbalance between the two provokes a state of oxidative stress. Among the major sources of ROS are mitochondria¹. Given their role in cellular respiration, they are responsible for the bulk of intracellular superoxide (O₂^•-) molecules². This mostly results from electron leakage to O₂ at complex 1 of the electron transport chain under conditions of strong negative inner mitochondrial membrane potential (Δψ_m), i.e., mitochondrial hyperpolarization. On the other hand, mitochondrial depolarization has also been correlated with increased ROS production pointing to multiple modes of action^3,4,5,6,7,8. Furthermore, through redox modifications in proteins of the fission-fusion machinery, ROS co-regulate mitochondrial morphology⁹.For example, fragmentation is correlated with increased ROS production and apoptosis^10,11, while filamentous mitochondria have been linked to nutrient starvation and protection against mitophagy¹². Given the intricate relationship between cellular ROS and mitochondrial morphofunction, both should ideally be quantified simultaneously in living cells. To do exactly this, a high-content imaging assay was developed based on automated widefield microscopy and image analysis of adherent cell cultures stained with the fluorescent probes CM-H₂DCFDA (ROS) and TMRM (mitochondrial Δψ_m and morphology). High-content imaging refers to the extraction of spatiotemporally rich (i.e., large number of descriptive features) information about cellular phenotypes using multiple complementary markers and automated image analyses. When combined with automated microscopy many samples can be screened in parallel (i.e. high-throughput), thereby increasing the statistical power of the assay. Indeed, a main asset of the protocol is that it allows for simultaneous quantification of multiple parameters in the same cell, and this for a large number of cells and conditions.

The protocol is divided into 8 parts (described in detail in the protocol below): 1) Seeding cells in a 96-well plate; 2) Preparation of stock solutions, working solutions and imaging buffer; 3) Setting up of the microscope; 4) Loading of the cells with CM-H₂DCFDA and TMRM; 5) First live imaging round to measure basal ROS levels and mitochondrial morphofunction; 6) Second live imaging round after addition of tert-butyl peroxide (TBHP) to measure induced ROS levels; 7) Automated image analysis; 8) Data Analysis, Quality Control and Visualization.

The assay was originally developed for normal human dermal fibroblasts (NHDF). Since these cells are large and flat, they are well suited for assessing mitochondrial morphology in 2D widefield images^13,14. However, with minor modifications, this method is applicable to other adherent cell types. Moreover, next to the combination of CM-H₂DCFDA and TMRM, the workflow complies with a variety of fluorescent dye pairs with different molecular specificities^1,15.

Protocol

The protocol below is described as performed for NHDF cells and with use of the multiwell plates specified in the materials file. See Figure 1 for a general overview of the workflow.

1. Preparation of Reagents

1. Prepare complete medium by supplementing Dulbecco's Modified Eagle Medium (DMEM) with 10% v/v Fetal Bovine Serum (FBS) and 100 IU/mL penicillin and 100 IU/mL streptomycin (PS). For 500 mL complete medium, add 50 mL of FBS and 5 mL of PS to 445 mL of DMEM.
2. Prepare HBSS-HEPES (HH) imaging buffer by supplementing Hank's Balanced Salt Solution (with magnesium and calcium, but without phenol red) with 20 mM HEPES. For 500 mL HH, add 10 mL of 1M HEPES stock solution to 490 mL HBSS. Verify the pH and, if necessary, adjust to pH 7.2.
3. Prepare 1 mM CM-H₂DCFDA stock solution by dissolving 50 µg of CM-H₂DCFDA lyophilized powder in 86.5 µL anhydrous DMSO. Mix by vortexing or pipetting up and down and make 20 µL aliquots in brown microcentrifuge tubes. Store in the dark at -20 °C and use within 1 week. If stored under N₂-atmosphere shelf life can be increased up to at least 1 month.
   1. Prepare 1 mM TMRM stock solution by dissolving 25 mg TMRM powder in 50 mL anhydrous DMSO. Mix by vortexing or pipetting up and down and make 10 µL aliquots in brown microcentrifuge tubes. Store in the dark at -20 °C. This solution is stable for at least a year.
4. Prepare a fresh aliquot of Tert-butyl peroxide (TBHP) stock (~ 7 M) for every experiment by directly pipetting a volume from the 70% stock solution (10 µL/96-well plate). Keep this aliquot at 4 °C until further use.
5. Prepare antibody working solution (AB) by diluting two secondary antibodies (Alexa Fluor 488 donkey anti-rabbit and CY3 donkey anti-rabbit) 1,000 times in 1x HH-buffer.

2. Setting Up of the Microscope and Acquisition Protocol ( ± 15 min)

NOTE: Image acquisition is performed with a wide-field microscope equipped with an automated stage and shutters, and a hardware based autofocus system using a 20X air Plan-corrected objective (NA = 0.75) and an EM-CCD camera. When setting up the assay for the first time, a test plate containing control cells, stained according to the protocol's instructions is used to calibrate the XY-stage and to optimize the acquisition settings. If the acquisition settings have already been determined, calibration can be done using an empty plate.

1. Lower the objective to the absolute base level and calibrate the XY-stage following software instructions.
2. Make sure the correct filter cubes are installed. To visualize CM-H₂DCFDA, use a standard GFP filter cube with a 472/30 nm excitation bandpass filter, a 495 nm cutoff dichroic mirror and a 520/35 nm bandpass emission filter. For TMRM, use a filter cube for TRITC with a 540/25 nm bandpass excitation filter, a 565 nm cutoff dichroic mirror and a 605/55 nm bandpass emission filter.
3. Create an imaging protocol using the acquisition software.
   1. Select the correct type of multiwell plate (manufacturer and code) from the list of available plates provided within the software. Alternatively define your own multiwell plate format using plate and well dimensions.
   2. Align the well plate according to the software's instructions, e.g. by defining two corners of the four outer corner wells. This step covers for camera orientation variation.
   3. Select the wells that need to be acquired. If this option is not available in the software, use a set of manually defined XY-locations that correspond to the selected wells.
   4. Optimize the acquisition settings (exposure time, lamp intensity, EM-gain) for the two channels separately using the test plate. Minimize exposure and intensity as fluorescence excitation light itself induces ROS. But, make sure the signal to background ratio is at least 2 for basal CM-H₂DCFDA and 3 for TMRM before TBHP treatment, and that there is no saturation after TBHP treatment. Acquisition settings greatly depend on the microscopy setup and cell type used, but as a reference, indicative settings when using a metal halide light bulb of 130 W as light source and NHDF cells stained according to the protocol's instructions are the following: for both CM-H₂DCFDA and TMRM an exposure time of 200 ms and ND filter 8 are used, combined with an EM-gain of 15 (13 MHz; 14-bit) and 4 (27 MHz; 14-bit) respectively. Once optimized for a certain setup and cell type, this step can be skipped.
      NOTE: it is essential that acquisition settings be kept the same throughout the entire imaging process. For large-scale, multi-day experiments, lamp stability should be warranted by regular quality control.
   5. Define an acquisition protocol, consisting of a sequential lambda (wavelength) acquisition. Select the CM-H₂DCFDA channel to be acquired first, to minimize light exposure before the measurement.
   6. Define a well-plate loop, to acquire 4 regularly spaced non-overlapping images positioned around the center of each well of the well selection using the acquisition protocol defined in 2.3.4. Choose meandering image acquisition, i.e., first from left to right, from well B02 to B11, then back, from right to left, from well C11 to C02 and so on (Figure 2A). This saves time compared to left-to-right image acquisition. If this option is not available in the software, adjust the custom set of XY-locations created in 2.3.3 to take on this imaging pattern.
   7. Save the XY-coordinates of the imaging-positions (e.g. in a separate xml-file), to allow easy revisiting in case of microscope recalibration. This is especially important if the readout from this assay has to be correlated with a post-hoc immunofluorescence (IF) staining for the same cells.

3. Seeding Cells in a 96-well Plate (45 - 90 min, Depending on the Number of Different Cell Lines)

1. Work in sterile environment such as a class 2 biosafety cabinet and wear gloves.
2. Decontaminate all surfaces and materials using 70% v/v ethanol in distilled water.
3. Take a cell culture flask with a 90% confluent cell culture from the incubator and place it in the biosafety cabinet.
4. Wash the cells twice with PBS 1x.
5. Add the appropriate amount of 0.05% trypsin-EDTA solution on the cells, making sure that the complete cell surface is covered (e.g., 1 mL for a T25 flask), and incubate for 2 min at 37 °C and 5% CO₂.
6. If all cells are detached (check with a microscope), add culture medium (DMEM + 10% FBS + 1% penicillin-streptomycin; ± 4 mL in a T25 flask) to inactivate the trypsin-EDTA solution.
7. Centrifuge for 5 min at 300 x g at room temperature.
8. Discard the supernatant and resuspend the cell pellet in culture medium. Determine the amount of medium for every cell type to obtain a cell concentration that is compatible with cell counting. Typically, a 90% confluent T25 flask of NHDF contains about 1-1.5 million cells, which are resuspended in 3-4 mL of culture medium.
9. Count the cells using a cell counting chamber or Coulter counter.
10. Seed 8,000-10,000 cells in each of the inner 60 wells of a black 96-well plate with a thin continuous polystyrene or glass bottom (black to avoid scattering and cross-talk between adjacent wells during imaging). When using different conditions/treatments/cell lines, distribute their seeding locations homogeneously on the plate so as to minimize plate effects (Figure 2A). The outer wells, except for well B01 and A01, are not used because they are more prone to edge effects.
11. Seed 8,000-10,000 cells in B01. This well will be used for focus adjustment, just prior to the image acquisition.
12. Fill the empty outer wells with medium to minimize gradients (temperature, humidity, etc.) between the wells and the environment.
13. Gently tap the plate three times before placing it back into the incubator to avoid cells from growing in patches.
14. Culture the cells for 24 h, or up to a confluence degree of approx. 70%.
15. Save the treatment information for the experiment into a spreadsheet called "Setup.xlsx". The file should contain four columns and will be used to link treatments with wells and image information during data analysis. The four columns are: 'Well', 'Treatmentnumber', 'Treatment' and 'Control' (one row per well). Every treatment is coupled with a unique treatment number, which is used during data visualization to determine the order of the treatments on the X-axis of plots. The control-column specifies the treatment that functions as control for the treatment on the current row. Illustrations of a typical experimental layout and corresponding setup file are depicted in Figure 2.

4. Loading of the Cells with CM-H₂DCFDA and TMRM ( ± 45 min)

NOTE: Handling of the cells on the day of the experiment can be carried out in a sterile environment (biosafety cabinet), but this is not mandatory because cells will be discarded or fixed directly after the assay.

1. Heat the HH-buffer to 37 °C.
2. Prepare a 20 µM TMRM working solution by diluting the 1 mM stock solution 50 times in HH-buffer (add 490 µL of HH-buffer to 1 aliquot of 10 µL TMRM stock solution).
3. Prepare a loading solution with 2 µM CM-H₂DCFDA and 100 nM TMRM. To this end, dilute the 1 mM CM-H₂DCFDA stock solution 500x and the 20 µM TMRM working solution 200x in HH-buffer.
   1. Typically, for 60 wells, prepare 7.5 mL of loading solution by adding 15 µL of CM-H₂DCFDA and 37.5 µL of TMRM solution to 7447.5 µL of HH.
4. Discard the culture medium from the cells by turning the plate upside down in a single fluid motion.
5. Gently wash the cells twice with HH-buffer using a multichannel pipette (100 µL/well). Discard the HH-buffer in between the washing steps by turning the plate upside down in a single fluid motion.
6. Load the cells with CM-H₂DCFDA and TMRM by adding 100 µL of the loading solution to each well, again using a multichannel pipette. Incubate for 25 min in the dark, at room temperature. Don’t forget well B01. 
7. During these 25 min, prepare working solutions of the oxidant TBHP and make sure the microscope and accessory hardware are turned on.
   1. Prepare working solution I: Dilute the 7M stock solution 70x to 100 mM (10 µL stock in 690 µL HH-buffer).
   2. Prepare working solution II: Dilute WS I 100x to 1 mM (10 µL WS I in 990 µL HH-buffer).
   3. Prepare working solution III: Dilute WS III 25x to 40 µM (for 60 wells add 300 µL WS II in 7200 µL HH buffer).
8. After 25 min, wash the cells again twice with 100 µL HH-buffer as described before.
9. Add 100 µL of HH-buffer to all 60 inner wells.

5. First Live Imaging Round to Measure Basal ROS Levels and Mitochondrial Morphofunction (± 15 min)

1. Make sure the acquisition software is operational and the imaging protocol is loaded.
2. Install the plate on the microscope, turn on the hardware based autofocus system and use well B01 to adjust the autofocus offset using the TMRM channel. As this procedure induces an increase in CM-H₂DCFDA signal intensity, this well is excluded from downstream image analysis.
3. Run the imaging protocol.

6. Second Live Imaging Round after Addition of TBHP to Measure Induced ROS Levels (± 20 min)

1. Carefully remove the 96-well plate from the microscope.
2. Add 100 µL of TBHP WS III (40 µM) to each well using a multichannel pipette (This results in a 20 µM TBHP concentration in the wells). This compound is used as an internal positive control for the CM-H₂DCFDA staining (the signal should rise) as well as a means to measure induced ROS levels.
   NOTE: H₂O₂ can also be used instead of TBHP, but this compound is less stable and therefore less reliable.
3. Wait at least 3 min to allow complete reaction of TBHP with CM-H₂DCFDA.
4. During this time, add 100 µL antibody working solution (1/1,000) to well A01.
5. Mount the plate back on the microscope and check focus again using well B01.
6. Acquire the same positions as in the first imaging round using the same imaging protocol.
7. Export the acquired datasets in a single folder as individual tiff files using standardized nomenclature that includes reference to the plate, pre- or post-TBHP treatment, well, field and channel, separated by underscores, e.g. 'P01_Pre_B02_0001_C1' for plate 1, pre-TBHP treatment, well B02, field 1 and channel 1. This information will be used during image analysis (e.g. to select the appropriate segmentation settings), as well as during data analysis (to connect analysis data with the correct treatments).
8. Acquire flat field images for both channels on all four positions around the center of well A01 using the acquisition protocol. Save them as individual tiff files in the same folder as the other images using the following standardized nomenclature: 'P01_FF_A01_0001_C1' for plate 1, field 1 and channel 1. Make sure the signals are well within the dynamic range; in case of saturation, use a lower concentration of antibody working solution.
9. Discard the plate or save for further processing.
   NOTE: Instead of removing the plate from the microscope and using a multichannel pipette to add the TBHP solution, an automated pipette can be installed on the microscope stage and connected with the acquisition software so as to function upon receiving a trigger. This allows for adding TBHP to every well directly after the first acquisition, before moving to the next well. This way, when the first imaging round is finished, the second one can start right away and all wells will have had an equal incubation time with the TBHP.

7. Image Processing and Analysis (± 30 min per 96-well plate)

NOTE: All image processing is performed in FIJI (http://fiji.sc), a packaged version of ImageJ freeware. A dedicated script was written for automated analysis of intracellular ROS- and mitochondrial signals, as well as morphological parameters (RedoxMetrics.ijm, available upon request). The underlying algorithms are described in Sieprath et al.¹.

1. Make sure FIJI is installed and operational.
2. Start up FIJI and install the macro-set (Plugins - > Macros - > Install …). This will invoke a number of new macro commands as well as a set of action tools to optimize the analysis settings, as shown in Figure 3A.
3. Open the setup interface to set the analysis settings by clicking on the 'S' button (Figure 3B).
   1. Select the image type, the number of channels and the well that was used for acquiring flatfield images.
   2. Indicate which channel contains cells (CM-H₂DCFDA channel) or mitochondria (TMRM-channel) and adjust the pre-processing and segmentation parameters for each channel depending on the image quality (Figure 3B). Tick the 'Background' and 'Contrast' checkboxes to perform background subtraction or contrast limited adaptive histogram equalization¹⁶, respectively. Define a sigma for Gaussian blurring of cells and Laplacian enhancement of mitochondria. Select an automatic thresholding algorithm and fill in the size exclusion limits (in pixels). If a fixed threshold is chosen instead of an automatic thresholding algorithm, fill in the upper threshold.
   3. Test the segmentation settings on a few selected images of the acquired data sets by opening them and clicking the 'C' or 'M' buttons in the menu for cell- or mitochondria segmentation respectively, and adjust the settings if necessary. An example result is shown in Figure 3C.
4. Run the batch analysis on the folder(s) of interest by clicking on the '#' button and selecting the folder with the images. Per folder, this will produce a new 'Output' directory, containing individual ROI sets (zip files) and result files (.txt) per image. For both channels the results file contains intensity and morphological descriptors. The CM-H₂DCFDA channel (cells) results file contains per descriptor the average value for the combined ROIs within one image. For the TMRM channel, the results file contains per descriptor the value for every individually segmented mitochondrial ROI.
5. After batch analysis, visually verify the segmentation performance on a 'Verification stack', a hyperstack of all images with their respective ROI overlay by clicking on the 'V' button. This way, artifacts such as over-/undersegmentation, out of focus images or dust-particles/fibers in images can already be spotted quickly. Further curation can be done during data quality control (cfr. §8).

8. Data Analysis, Quality Control (QC) and Visualization

Processing and analysis of the raw data is done using R statistical freeware (http://www.rproject.org – version 3.3.2) and RStudio (http://www.rstudio.com/ – version 1.0.44). To quickly obtain and visualize the results, an intuitive Shiny application¹⁷ (available upon request) has been conceived that integrates and visualizes the data in heatmaps and boxplots, and also performs statistical analyses. In general, the workflow comprises of two consecutive steps. First, data is processed and inspected per 96-well plate to detect aberrant data points. Secondly, curated data from all plates of a given experiment are combined and analyzed using non-parametric multivariate tests¹⁸ and a principal component analysis.

1. Make sure R and RStudio are installed and operational.
2. Start RStudio.
3. Open the RedoxMetrics shiny application and run the app (choose to run it in an external browser).
4. On the 'input' page, select the directory where the results files from RedoxMetrics.ijm are located.
5. Make sure 'Setup.xlsx' (created in step 3.15) is also present inside this directory.
6. The results files and setup information are automatically imported, rearranged and visualized.
   1. The 'experimental setup' page shows the layout of the experiment. Use this page for verification.
   2. The next page, 'results per plate' shows the data for each plate separately in a color-coded multi-well plate layout as well as in boxplots with outliers labeled with well name and image number. The latter allows facile inspection and identification of aberrant data points (extremely high or low values compared to the average values measured for that specific treatment - see Figure 4 for an example).
      Using this information, verify the images corresponding to the aberrant points. If abnormalities are detected, they have to be removed from the analysis. Most abnormalities are caused by improper segmentation when (part) of the image is out of focus, or when highly fluorescent dust particles disturb proper segmentation. Extreme cases can already be spotted quickly using the verification stack tool (step 7.5), but more subtle occurrences are discovered at this step. When no apparent or technical reason can be found for the aberrant value, the image should not be removed from analysis.
   3. Optional: create a new spreadsheet called 'Drop.xlsx' with only one column called 'Drop'. In this column, list the file names (including ".tif" extension) of all the images that have to be removed from the analysis (identified in step 7.5 or step 8.6.2). Upload this file using the 'input' page.
   4. The 'results whole experiment' page shows the results from all plates combined. If a drop file was uploaded, this file is used to remove the specified data points from the analysis.
      For each parameter individually, the data are normalized per plate according to their respective controls. Data from all plates are then combined, followed by non-parametric multivariate tests from the nparcomp package¹⁸. If only 2 treatments are compared two sample tests for the nonparametric Behrens-Fisher problem are performed. For more than 2 treatments, a non-parametric contrast-based multiple comparison test is used. The results are visualized using boxplots.
   5. The 'cluster analysis' page shows the results of a principal component analysis (PCA - R core 'stats' package). Data from 5 parameters (basal & induced ROS, and mitochondrial membrane potential, size & circularity) are combined to discriminate the different treatments based on a sensitive redox profile. To this end, a principal component analysis (PCA) is performed (R core 'stats' package). The results are visualized using a biplot (ggbiplot package -  http://github.com/vqv/ggbiplot).
   6. Use the 'download data' page to download the backend data frames containing the processed and rearranged data such that they can be reused for more advanced data visualization or statistical analyses.
      NOTE: Typical pitfalls and potential solutions are listed in Table 1.

Results

The assay has been benchmarked using several control experiments, the results of which are described in Sieprath et al.¹. In brief, the fluorescence response of CM-H₂DCFDA and TMRM to extraneously induced changes in intracellular ROS and Δψ_m, respectively has been quantified to determine the dynamic range. For CM-H₂DCFDA, NHDF showed a linear increase in fluorescence signal when treated with increasing concentrati...

Discussion

This paper describes a high-content microscopy method for the simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction in NHDF. Its performance was demonstrated with a case study on SQV-treated NHDF. The results support earlier evidence from literature in which increased ROS levels or mitochondrial dysfunction have been observed after treatment with type 1 HIV protease inhibitors, albeit in separate experiments^19,20,

Materials

Name	Company	Catalog Number	Comments
Reagents
Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)	ThermoFisher Scientific	T668
CM-H2DCFDA (General Oxidative Stress Indicator)	ThermoFisher Scientific	C6827
Dimethyl sulfoxide	Sigma)Aldrich	D8418
MatriPlate 96-Well Glass Bottom MicroWell Plate 630 µL-Black 0.17 mm Low Glass Lidded	Brooks life science systems	MGB096-1-2-LG-L
HBSS w/o Phenol Red 500 mL	Lonza	BE10-527F
DMEM high glucose with L-glutamine	Lonza	BE12-604F
Phosphate Bufered Saline (PBS) w/o Ca and Mg	Lonza	BE17-516F
HEPES 1 M 500 mL	Lonza	17-737F
Trypsin-Versene (EDTA) Solution	Lonza	BE17-161E
Cy3 AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson	711-166-152	Antibody used for acquiring flat-field image
Alexa Fluor 488 AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson	711-546-152	Antibody used for acquiring flat-field image
Name	Company	Catalog Number	Comments
Equipment
Nikon Ti eclipse widefield microscope	Nikon
Perfect Focus System (PFS)	Nikon		hardware-based autofocus system
CFI Plan Apo Lambda 20X objective	Nikon
Name	Company	Catalog Number	Comments
Software
NIS Elelements Advanced Research 4.5 with JOBS module	Nikon		This software is used to steer the microscope and program/perform the automatic image acquisition prototocol
ImageJ (FIJI) Version 2.0.0-rc-43/1.50g
RStudio Version 1.0.44	Rstudio
R version 3.3.2