Yan Lab by Yasir Akhtar, Lindsey Myers, Zhen Yan July 22, Skeletal Muscle Fiber-TMRM Assay.v2.5

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1 Skeletal Muscle Fiber-TMRM Assay.v2.5 The assay, based on the methods published previously 1-2, will measure the susceptibility of isolated myofibers to the ATPase inhibitor, oligomycin. The cationic tramethyl rhodamine methyl ester (TMRM) accumulates in polarized mitochondria and is released when Δψ m decreases. Mitochondrial permeability transition leads to loss of Δψ m and subsequent loss of the TMRM signal. Hence, this assay will indirectly measure MPT in mitochondrial population as a whole. FCCP is added at the end to confirm that what we are measuring is indeed due to decrease in Δψ m. Isolation of myofibers from FDB muscle 1. Take a laminin coated plate (see below for the coating procedure) and wash with PBS twice. Add 1 ml of DMEM, 10% FBS and incubate at 37ºC and 5% CO2 until the muscle fibers are ready for plating. 2. Sacrifice mice by cervical dislocation under anesthesia. Place the mice on a sterile paper towel. 3. Clean the foot and lower hind limb with betadine once and 70% alcohol twice using sterile gauze. EVERYTHING FROM NOW ON HAS TO BE UNDER STERILE CONDITIONS 4. Cut the foot including the ankle using a pair of sterile scissors without damaging the tendons. 5. Place the foot on a sterile paper towel-covered wooden panel under the dissection microscope with two 27-gauge needles pinning through the 1 st and 5 th digits to hold it down with the plantar side upwards. 6. Use a pair of sterile scissors to cut the skin open on both sides of the foot longitudinally to the digits. Lift up the skin and remove it by cutting the distal connection to the digits. FDB muscle with three tendons should be visible. Pipette some normal saline to avoid drying up. 7. Grab the proximal tendon gently with a pair of thin hemostats and cut the tendon. Gently lift up FDB and cut the connective tissue under the muscle to the three tendons attaching to the 3 middle digits. Cut them and remove FDB. 8. Place the muscle in 1 ml of collagenase solution (0.2% Type-II collagenase, 0.2% BSA in Tyrode s buffer with PenStrep) in a 35-mm tissue culture plate (placed in another plate) and digest at 4ºC for 1 hr in the refrigerator (or a cold room). Place on the rocking table table and set at level Gently shake the plate and digest for 2 hr at 37ºC in a tissue culture incubator. Gently shake the plate every 30 min. 10. Pick up the muscle fibers by a plastic Pasteur pipette from the plate and transfer the muscle fibers to 1 ml culture medium (DMEM, 10% FBS) in a 35-mm plate. 11. Disperse the muscle fibers by passing them gently through a wide-pore plastic Pasteur pipette 30 times to release single fibers. Do not make air bubbles. 12. Place half of the mixture of dispersed myofibers into a laminin coated plate and the other half to another well. Add about 0.5 ml of culture medium. 13. Incubate at 37ºC and 5% CO 2 for up to 24 hrs before the assay.

2 TMRM assay 1. Wash the attached muscle fibers with 1 ml of pre-warmed Tyrode s solution, and remove the solution. 2. Add 1 ml of 20 nm TMRM in Tyrode s solution and incubate at 37 C for 20 min in the tissue culture incubator. Protect the samples from lights. 3. Wash the cells with 1 ml of pre-warmed 1X Tyrode s solution 2 times. 4. Add 1 ml of tyrodes to the well. 5. Borrow the key for the microscope room/ideas Lab from Pam (Wamhoff lab) 6. Get access to MR5 2 nd floor on your ID tag. Take sample to MR5, 2 nd Floor to microscopy suite (Room number 2208). 7. You will need your sample (covered with aluminum), a Timer, a USB drive, Tyrode s, PBS, 1 ml and 10-µl pipette with tips to take to MR5. Also make sure to bring Oligomycin/FCCP working stocks on ice. 8. Turn on the fluorescent and 2 camera switches below the desk (3 switches to turn) 9. Turn on the computer and log in to windows through eservices and open the software IPLab Visualize your cells with transmission light after setting optic to x10. The lamp will need to be ON to visualize under transmission light. Turn off the lamp after visualizing. (If the lamp stays on, fluorescence won t be visualize) 11. Click on OPEN SHUTTER in the program and choose the emission wavelength CYP (orange). Visualize under fluorescence. Emission shutter 2 should be selected (below the lens) 12. Make sure to follow the settings: Go to camera, acquire 13. Under the tab TL Stream set the following preferences: a. Under timelapse change the drop down box Interval & Exp. Length b. Interval should be set 1 and the drop down box to min c. Experimental Length should be set to 50 and the drop down box to min d. Z frames should be set to 1 e. Check the box grab to disk f. Check use Window Name g. File path: browse to your thumb drive and create a new folder for each mouse h. In the drop down box save pictures as IPLab as the file type i. Streaming should not be selected 14. Under the tab General set the following preferences: a. The preview box should be checked b. Image Destination should be selected as new and then named TMRM _ Mouse # _ DATE c. In the drop down box: Process: No processing d. Create Timing Window and Display after Each Grab should be selected e. 1 Red or [B\W] should be selected 15. Under the tab Exp. a. Exposure Time [in ms]: will vary and should not be messed with until after all the setting are preset. b. Auto-Exposure i. Target Max Val: 2048 ii. None should be selected

3 16. Click Okay at the bottom of the window 17. An additional window with the live preview screen will appear, adjust the Contrast settings to make the fiber stand out. Click the button to begin picture sequence. 18. Measure baseline fluorescence 19. After the first picture is taken add 100 µl of Oligomycin (100 µm) to give a final concentration of 10 µm, and titrate a few times to ensure mixing 20. Add 100 µl of PBS in the control well (Oligomycin is dissolved in PBS) 21. After the 40 minute mark picture is taken add 100 µl of FCCP (100 µm) final concentration of 10 µm, and titrate a few times to ensure mixing Image analysis 1. Open ImageJ. You can download it for free on the internet if you do not already own a copy 2. Open the folder containing the files on the desktop. 3. Select the files as a whole. This can be done by selecting the first in the sequence, holding down shift and selecting the last in the series. 4. Drag the selected files to the ImageJ window and drop them this will cause all the selected files to open on your desktop. 5. Starting with you initial picture in the sequence. Select the heart shaped button, this will allow you to free hand circle the fiber. 6. Once you have completely circled the fiber be careful not to click on the screen as this will null the action of circling. 7. On the program window select edit:selection:fit spine. This will allow you to carefully adjust the parameters in case there was a small error. 8. Then click Analyze:Set Measurments. Check Area and Intergrated Density. Then press OK 9. Select the top of the window, being careful not to select inside the window. 10. Hit Command+M. This will take the measurement of the selected area. 11. Then follow the sequence of Command+C to copy 12. Select the next picture in the sequence. Press Control+V to paste and then Control+Z to remove the fiber from the previous pane. This will leave the spine on the image and you can control the placement with the arrow function keys. Press Control+M when you are ready to take the measurement 13. Do this for all pictures in your sequence. Once you have copied the spine, it will be set for all pictures and you only need to hit Control+V and Control+Z and then place the spine on the fiber for subsequent photos and then take the measurement by Control+M. 14. Once you have achieved all the measurements for your set of data. On the measurement window go to Edit: Select all and then Control+C to copy the data. 15. Paste the data into excel and then it is capable of quantifying. Reagents Laminin solution and coating of the plate 1. Store the original laminin stock (Sigma L2020, 1 mg/ml) at -80 C. 2. Thaw the laminin stock on ice for 60 min prior to coating the plate. It is extremely important to thaw it slowly. Quick thaw will cause gel formation and lose coating capability.

4 3. Add ice-cold sterile phosphate buffered saline (PBS), 1X (CaCl2/MgCl2-free) to the laminin stock for a final concentration of 15 µg/ml. 4. Add 0.2 ml of laminin to cover the bottom of each 48-well plate well, and incubate at 37 C for 2 hr. Coat 1 well for each plate and label them. 5. Remove the laminin solution and air-dry the plates at room temperature under UV light in the tissue culture hood for 30 min. 6. Wrap and store the plates in a cool place. Tyrode s solution (1 Liter) Reagent Source Catalog# MW/Conc. Quantity Final Conc. NaCl 2.5 M Fisher F ml 135 mm KCl 1 M Fisher P ml 4 mm CaCl 2.2H 2 O 1M Acros ml 1 mm MgCl 2.6H 2 O 1M Fisher M ml 1 mm KH 2 PO 4 1M Fisher P µl 0.33 mm Glucose 1M MP ml 10 mm HEPES 1M 10 ml 10 mm Collagenase digestion solution 1. Prepare collagenase digestion solution fresh before use. 2. Take the collagenase (Collagenase Type II, CLS2 Worthington Lab) from 4 C in a desciccator. Warm up in your hand completely before opening it. 3. Weigh certain amount of collagenase using a wax paper. (2 mg/ml. 1 ml will be enough for 1 FDB muscle) 4. Pour the collagenase into a 15-ml tube. 5. Add certain amount of Tyrode s solution to make a final concentration of collagenase 0.2% (2 mg/ml). 6. Add fatty acid free BSA for a final concentration of 0.2%. 7. For example, to make 5 ml of solution, add 10 mg of collagenase and 10 mg of BSA and top off the volume to 5 ml with Tyrode s buffer. Filer and add 50 ul of PenStrep. 8. Filter sterilize the solution by using a 22-micron filter. 9. Store the solution on ice. TMRM 1. Store the stock solution (1 mm) in 5 µl-aliquots in -20ºC (Box 23 at the bottom). 0.5 mg/ml is equivalent to 1 mm. 2. Add 2 µl of the stock to 98 µl of 1X Tyrodes buffer (1:50 dilution) to dilute it to 20 µm. 3. Further dilute it by adding 2 µl it to 2 ml of 1X Tyrodes buffer to 20 nm (1:1000 dilution). AVOID EXPOSURE TO LIGHT DURING HANDLING FCCP 10 mm Sigma C2920 MW Dissolve in FCCP in DMSO (or Ethanol/Acetone) to 100 mm stock (santacruz). Further dilute with ddh20 into alliquots of 100 µm is in the -20ºC in boxes on the shelf.

5 Oligomycin 1 mm Sigma O4876 MW Dissolve in DMSO( or Acetone/Ethanol) (cellsignal) to a stock concentration of 1 M Soluble in DMSO (300mg/ml), 100% ethanol (200mg/ml) or acetone (800mg/ml)- Enzolife Sciences Further dissolve in ddh20 to make working stock of 100 um. Reference 1. Bupivicaine Myotoxicity Is Mediated by Mitochondria 2. Genetic Ablation of Cyclophilin D rescues mitochondrial defects and prevents muscle apoptosis in collagen VI myopathic mice