Skinned Myocyte
Skinned Myocyte Protocol (updated: 12.2.16)
Myocyte Isolation
- Add to Isolation Solution: DTT (1:100 1M), protease inhibitor (1:100), phosphatase inhibitor, Triton X-100 (0.3%, 30uL 10% solution into 1mL of solution, keep 9ml without Triton)
- Add to Relaxing solution and Activating Solution
1. Get tissue from liquid N2 storage or in -80. Keep samples on liquid N2
2. Cut a small sample of heart tissue (~10mg)
3. Place sample in 1 mL Isolation Solution with triton in glass culture tube
4. Homogenise in large homogenizer speed of 7500 for one second three times. This should be enough to homogenise the tissue but you can homogenise for an extra one second if needed.
5. Transfer to a Falcon tube using a cell strainer filter (run the solution through the filter)
6. Incubate for 20 mins on ice (clean homogenizer during this step)
7. Centrifuge solution at 120g for 2 mins in the large centrifuge (4° C)
8. Remove supernatant
9. Resuspend the pellet in 1 ml Isolation Solution without triton
10. Transfer to an Eppendorf tube
11. Repeat steps 7-9 (washes with Isolation Solution no triton) for two more times
Myocyte Preparation
1. Turn on computer. Power up force transducer >1 h before force is going to be measured, to give it time to warm up. Turn on length controller, turn on light power source. Turn on needle manipulators
2. Arrange needles with naked eye, make sure that you can see them with a 4x objective, then bring them straight up
3. Take 40 uL of sample from middle/ lower upper portion of the tube and place in center of a 22mm coverslip
4. Check that density of cells is good with 4x objective
5. Using the 10x objective, find a good myocyte. Long, not too thin/thick, nothing attached to it. Cell surface should be smooth, not bumpy. Increase magnification to 40x with HMC and check for good sarcomere pattern.
6. The coverslip may need to be rotated so that the myocyte is oriented horizontally
7. Go back to 4x and put needles down (max speed is okay, but keep checking with eye to make sure they don’t hit the stage)
8. Go to 10x and slow down movement (Medium speed is OK). Get needles closer
9. Position needles over the myocyte on the 10 X objective, but not attach
10. Bring needles straight up, out of the drop
11. Put a drop of Norland Optical Adhesive 63.
12. Move the stage only horizontally. Move so the drop of glue goes through the middle of the field of view
13. Bring down one needle. Keep changing focal plane to make sure the needle is below the top of the glue, but not hitting the coverslip
14. Move stage left and right a couple times to coat needle with glue
15. Bring needle up a little bit, and then straight down into the glue to cover the tip
16. Lift needle straight up
17. Repeat for second needle
18. Make sure both needles are high enough to clear the drop of sample
19. Move stage horizontally and find original myocyte
20. Open VSL program on computer
21. Go to 40 x objective and make sure that the myocyte is in the middle of the screen and the SL is clear
22. On 10x, bring down needles until they are close to the myocyte on Medium speed
23. Go back to 40x, now you will be able to see the myocyte and the needles on the screen. If the needles are not visible under the 40x objective, go back to 10x and re-position needles again.
24. Turn speed of needles to slow (fast or medium range is OK)
25. Bring down one needle until it slightly squishes the myocyte.
26. Move needle slightly in y-direction (rolls the myocyte in glue) - OPTIONAL
27. Repeat for second needle
28. Lift up a little the left needle, to make sure that the myocyte comes up with the needle, and then repeat with the right needle. If myocyte moves with needles, it means it attached
29. Move both needles up, to move myocyte off the coverslip but not out the solution so that it doesn’t dry out. Careful to keep needles on the same level.
30. Set glue with UV lamp (3x10 seconds – one to left, one to right, one from below)
Force-Calcium Experiment
1. Using the VSL program, slowly move the needles so that the SL of the myocyte is stretched to 2.1
2. Fill small bath with relaxing solution (140 µL). Line up stage so that one short movement will move myocyte into relaxing solution. Move myocyte and pins from isolation solution to relax solution.
3. Open the ASI program
4. Go to Scope, and then open
5. Then go to Setup, and then Open.
6. Press Zero Lout and put the length of the myocyte. Press Record Lin. At the bottom it should say that the reference length was changed to [your value]
7. To make sure that the length controller is working, press Freeze time on the Scope tab. You should see the force of the myocyte in real time.
8. Press Start test on ASI program while in relaxing solution. The length controller slacks the myocyte for 10 seconds and then re-lenghten again.
9. To save the data, press Open Log File on the Scope tab. Select unique name and then press start on the Scope tab. If you don’t press start then your data wont be saved!
10. Put 140 uL of Activating solution onto the second small bath.
11. Move the myocyte to the activating solution. You should see it contracting and the force on the Scope tab should be rising.
12. When the force reaches a plateau, press Start test on the ASI tab and then quickly move the myocyte back to the relaxing solution.
13. Press Freeze time on the Scope tab, this stops the real time of the force which helps you to record your data
14. On your lab notebook, write down the higher (SL of 2.1) and lower (slack) force values of the myocyte, for both activating and relaxing solution.
- The difference in these numbers in activation solution is the Total force and the one in relaxing solution is the passive force. Active force= Total force - passive force.
15. Make the rest of activating solutions by mixing together activating and relaxing solution: 90%, 85%, 80%, 75%, 65%.
16. Add 140 uL of each solution to the small baths
17. Repeat force measurements for each solution
18. At the end, do another 100% activating solution to measure rundown.
19. Measure width of myocyte using calibrated camera (RECORD)
20. Measure depth of the myocyte using a mirror, or calibrated manual focus knob (RECORD)
Clean up
1. Let tips sit in 5M NaOH for 10 minutes (10 um drop)
2. Let tips sit in H2O for 5 minutes (10ul drop)
3. Let tips sit in 100% EtOH for 5 minutes or until it evaporates (10ul drop).
4. Make sure that pins are clean by looking at them at the 40x objective
Shut down for day
1. Move pins up from stage. Move away from each other.
2. Turn off force transducer, length controller, light bulb, pin manipulators and computer (if you do experiments every day you can leave computer on, but turn off at the end of the week)
