ViaFuse: Fiji macros to calculate skeletal muscle cell viability and fusion index

C2C12 cell culture

Cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum, 2 mM l-glutamine, and 0.01% penicillin and streptomycin. When cells reached 90% confluency, the media was switched to differentiation media which was composed of DMEM, 2% horse serum, 2 mM l-glutamine, and 0.01% penicillin and streptomycin.

Delivery of small interfering RNAs (si-RNAs) via electroporation

C2C12 myoblast cells were transfected with two different Capzb si-RNAs from Dharmacon (si-Capzb-#1: #MQ-040494-01-0002 and si-Capzb-#2: #MQ-040494-01-0004) or the si-GENOME negative control also from Dharmacon (#D-001210-01-05), all at a final concentration of 87 pmol/mL. The Neon electroporation system (Thermo Fisher Scientific, MPK10096) was used to deliver the si-RNAs into C2C12 myoblasts according to the manufacturer instructions: 750,000 cells per well were electroporated using a 100 μL Neon pipette with conditions of 1650 V, width of 3 ms, and three pulses. The next day (24 h after electroporation), myoblasts were induced to differentiate into myotubes. After 4 days of differentiation, protein lysates were extracted from the cells or the cells were fixed, immunostained, and visualized by confocal microscopy.

Protein lysate preparation

Cells were placed on ice, washed with cold PBS, and lysed using ice cold RIPA buffer (50 mM Tris, 120 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate) containing protease (Roche, #11873580001) and phosphatase (Roche, #04906845001) inhibitors. Lysates were incubated on ice for 15 min, then sonicated at 75% amplitude (intensity) for 3 min (30 s on and 30 s off). The lysates were incubated on ice for 15 min and then centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatants were transferred into new tubes and stored at − 80 °C. Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific, #23225).

Western blotting assays

Protein lysates (30 μg) were analyzed by western blotting to verify efficient CAPZB depletion. Samples were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% mini-Protean TGX stain-free gel (BioRad, #4568044). The running buffer consisted of 200 mM glycine, 25 mM Tris base, and 3.5 mM SDS. Electrophoresis was performed at 90 V for 30 min and then 150 V for another 30 min. Gels were imaged using the ChemiDoc Imaging System from BioRad. The proteins were then transferred from the gel to an Amersham Hybond Low Fluorescence 0.2 μm PVDF membrane (GE Healthcare Life Sciences, #10600022) at 100 V for 1 h in transfer buffer (100 mM glycine, 25 mM Tris, and 20% methanol). Total proteins in the membranes were visualized using the ChemiDoc Imaging System from BioRad. Membranes were then blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline buffer containing tween 20 (TBST) (400 mM Tris base, 2.74 M NaCl, 0.1% tween 20, pH 7.6). Membranes were briefly washed with TBST and then incubated with the appropriate primary antibodies diluted in TBST overnight at 4 °C: mouse anti-LMNA (Santa Cruz Biotechnologies, sc-7293, dilution 1:500), rabbit anti-CAPZB (Abcam, ab220669, dilution 1:100). The following day, membranes were washed with TBST three times (10 min each) and then incubated for 1 h with the secondary goat anti-mouse antibody Dylight 800 from Thermo Fisher Scientific (SA5–35521, dilution 1:10,000). Membranes were imaged using an Odyssey LiCor Imager.

Immunofluorescence assays

On the fourth day of differentiation, cells were fixed for 2 min in a 2% paraformaldehyde solution in cell medium (a 4% paraformaldehyde solution in PBS—made from EMS 16% Paraformaldehyde Aqueous Solution EM Grade, Fisher Scientific, #15710—was diluted in half with cell culture medium). The media containing the paraformaldehyde solution was aspirated, and cells were then incubated with 2% paraformaldehyde in PBS for 20 min. Cells were washed with PBS three times (10 min each) and then blocked in an immunofluorescence buffer (BIF) containing PBS, 1% bovine serum albumin, and 0.3% Triton for 1 h at room temperature. Cells were next incubated with the mouse polyclonal IgG anti-myosin heavy chain (MYH) 3 (Santa Cruz Biotechnologies, sc-53091) diluted (1:50) in BIF overnight at 4 °C. The following day, cells were washed with PBS three times (10 min each) and then incubated for 1 h in the darkness and at room temperature with an anti-mouse antibody conjugated with Alexa Fluor 488 (AlexaFluor 488((ab)) 2 fragment of goat anti-mouse IgG (HHL), Thermo Fisher Scientific, A1101) diluted (1:500) in BIF. Cells were then washed with PBS three times (10 min each) and incubated with 2 μM DAPI diluted in PBS for 5 min at room temperature. Cells were washed twice with PBS and imaged in PBS immediately.

Confocal microscopy

Cells were imaged in the Hooker Imaging Core at The University of North Carolina at Chapel Hill using a Zeiss LSM 880 confocal microscope with a 10x Plan Apochromat objective (0.45 WD) and the Zen Black (Zeiss) software was utilized for image acquisition. Stitched images (3 × 3) were taken of each condition. The following excitation parameters were used: argon multiline laser at 488 nm (for Alexa Fluor 488) and a 405 nm diode at 30 mW (for DAPI). Cells were imaged at room temperature.

Manual image analysis

The captured images were analyzed using Fiji software. The images were unstacked and DAPI was assigned a red color and MYH was assigned a green color. DAPI and MYH images were multiplied variably to ensure all images looked similar for quantification. To calculate the total number of nuclei, a threshold was first applied to the DAPI image to make it binary, small holes were then filled and a watershed separation was utilized to separate close nuclei. The area of nuclei sized between 50 μm2 and 250 μm2 and those greater than 251 μm2 was procured using the analyze particles tool. To determine the number of nuclei in nuclear clumps, the areas of the objects larger than 251 μm2 were divided by the mode of the nuclei areas between 50 μm2 and 250 μm2. The obtained number of nuclei in nuclear clumps was then added to the number of nuclei with areas between 50 μm2 and 250 μm2 to generate the total number of nuclei in an image. Finally, the total number of nuclei in the image was divided by the image size (in mm2) to procure the total number of nuclei per unit area (nuclei/mm2). For calculation of the fusion index, the DAPI and MYH images were merged. Using the grid and count cells plugin, the nuclei that were found outside of MYH-positive cells and those in MYH-positive cells with one or two nuclei were counted. Fusion index was thus calculated using the following equation:

$$\mathrm\ \mathrm\mathrm=\frac\ \mathrm-\mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm-\mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm\ 1\ \mathrm\ 2\ \mathrm}\ \mathrm}\times 100$$

Operation and guidelines to install ViaFuse macros

To run the ViaFuse macros, the computer must be a Windows XP or later, a Mac OS X 10.8 “Mountain Lion” or later, or a Linux on amd64 and × 86 architectures. The system must also run Java 8 or later. To install Fiji, the following steps should be followed: (1) navigate to https://Fiji.sc/, (2) scroll to the “Downloads” section and download the correct version of Fiji for the computer (the following macros were made with version 1.53c for MacOS and version 1.53 h for Windows), and (3) follow Fiji installation instructions and ensure that the correct version is installed.

To download the ViaFuse macros, two steps should be followed: (1) navigate to https://github.com/tasneemsmacros/ViaFuse and (2) download the desired macros (ViaFuse-V or ViaFuse-F) for your operating system and save them to your computer. Instructions for each macro are available for download at that link as well.

ViaFuse-V, macros to calculate total nuclei

ViaFuse-V uses the DAPI image to generate a .csv file with (i) a list of the areas of particles with sizes greater than 251 μm2, (ii) the number of nuclei sized between 50 μm2 and 250 μm2, (iii) the mode of nuclei sized between 50 μm2 and 250 μm2, and (iv) the area of the image (in mm2). To calculate the number of nuclei in clumps, the user divides the sizes of particles greater than 251 μm2 (generated by ViaFuse-V) by the mode of nuclei sized between 50 μm2 and 250 μm2 (generated by ViaFuse-V). This number can be added to the number of nuclei sized 50 μm2 and 250 μm2 (generated by ViaFuse-V) to produce the total nuclei number in an image. The total nuclei number is then divided by the image size measured in mm2 (generated by ViaFuse-V) to calculate the total number of nuclei per unit area (total nuclei/mm2).

To run the ViaFuse-V macro, open Fiji software and upload the desired image in “.lsm” format for stacked images or “.tif” format for unstacked images. If working with stacked images, unstack them (Image>Stacks>Stack to Images) and close all images except for the nuclei image. This is a crucial step to ensure the macro performs the analysis on the correct image. Run the macro on the image (Plugins>Macros>Run>Select Macro). The macro will prompt the user to select the folder where results will reside. Once the user has selected the desired folder, click “Open”. Another dialog box will appear. The first box will have the file path based on the previous selection. Click on the second box and type the desired name of the .csv file of the results. Click “OK” when finished. The program will then prompt the user for a multiplication value for the image being analyzed. A number will need to be typed and then click “OK” when done. The process will take a few minutes to finish. When the macro finishes the calculations, a “Calculations Complete!” message will be displayed. Click “OK” to exit the message. The results file can be found on the user’s computer where they chose to save it. This macro assumes that the units of the image are already in μm units. If this is not the case, the image needs to be converted accordingly. The numbers output from ViaFuse-V can be recorded into an excel that will generate the total number of nuclei (Supplementary Table 1).

ViaFuse-F, macros to calculate fusion index

ViaFuse-F creates a mask of the MYH image and subtracts it from the DAPI image to generate an outside nuclei image with the nuclei in MYH-positive cells removed. ViaFuse-F uses this outside nuclei image to generate a .csv file that has (i) a list of the areas of particles greater than 251 μm2, (ii) the number of nuclei with areas between 50 μm2 and 250 μm2, and (iii) the mode of the areas of nuclei between 50 μm2 and 250 μm2. The number of nuclei that are in nuclear clumps outside of MYH-positive cells is determined by taking the sizes of nuclei greater than 251 μm2 that are outside of MYH-positive cells (generated by ViaFuse-F) and dividing them by the mode of the area of nuclei in the outside nuclei image sized between 50 μm2 and 250 μm2 (generated by ViaFuse-F). The number of nuclei in MYH-positive cells with less than three nuclei needs to be counted by the user. The total nuclei number from ViaFuse-V is used in the final fusion index equation. Fusion index is thus calculated using the following equation:

$$\mathrm\ \mathrm\mathrm=\frac\ \mathrm-\mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm-\mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm\ \mathrm\ 1\ \mathrm\ 2\ \mathrm}\ \mathrm}\times 100$$

To run the ViaFuse-F macro, open Fiji and upload the image in “.lsm” format. Ensure that the DAPI and MYH images are stacked together. Do not unstack the images. If other images are open besides the DAPI and MYH images this will not impact the macro. The macro assumes a specific stack order that is specified in the macro instructions available at the download link. Run the macro on the image (Plugins>Macros>Run>Select Macro). There will be a prompt for the user to select the folder to deposit results. Once the user has selected the desired folder, click “Open” and a dialog box will appear. The first box will have the file path based on the previous selection. Click the second box and type the desired name of the .csv file of the results. Click “OK” when finished. The program will then ask the user for a multiplication value for both the MYH image and DAPI image. Type in a number in each box, then click “OK” when done. The process will take a few minutes to finish. When the macro finishes the calculations, a “Calculations Complete!” message will be displayed. Click “OK” to exit the message. The .csv file can be found on the user’s computer in the folder where the user chose to deposit results. The merged .png image of the myotubes and nuclei counted can be found in the same location with the same name as the .csv file.  The numbers output from ViaFuse-F together with the manually counted nuclei in myotubes with 1 or 2 nuclei can be recorded into an excel that will generate the fusion index (Supplementary Table 1).

Operation and guidelines for MyoCount usage

The images to be quantified using MyoCount were first divided into 3 × 3 montages using Fiji. The nine panels for each image were processed in a batch using the MyoCount program as previously described [11]. The following parameters were set for analysis: SmallestMyotubePixelCount = 200, SmallestNucleusPixelCount = 50, NucFillSize = 2, MaxCircleRad = 30–35, MaxNucSizeDivisor = 200, TubeThresh = 0.73.

Testing

Researchers quantified total number of nuclei per unit area (mm2) and the fusion index of nine images of immunofluorescence-stained myotubes from three experiments using Fiji/ImageJ software (version 1.53c on MacOS, version 1.53 h on Windows), MyoCount program (version 1.3.1 with MATLAB Runtime Version 9.4 R2018a on Windows), and the manual image analysis methods described above. For the ViaFuse macros quantification, the default settings of the macros were used, and the multiplication values of each image were adjusted by the investigators. A Pearson correlation coefficient was calculated to compare values from the ViaFuse to those achieved manually or through the usage of the MyoCount program. All data analysis was performed using Microsoft Excel.

Statistical analysis

Statistical analysis was performed in Microsoft Excel (Microsoft). Significance was determined using an unpaired Welch’s t test (two-tailed). The difference between two conditions was considered statistically significant when p < 0.05. All data are presented as the mean plus and minus the standard error from the mean (sem).

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