Download Adult mouse myocyte harvest protocol

Adult mouse myocyte harvest protocol
Protocol for adult mouse myocyte isolation edited from a protocol kindly provided by:
Dr. Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA
Procedure:
1. Prepare the following solutions. The base solution is Hanks Balanced Salt Solution. All solutions should be
filtered.
HBSS
2,3 Butandione
monoxime (BDM)
Glucose
Taurine
BSA
CaCl2
Perfusion Buffer
1000 ml
1.01 g (10 mM)
Buffer A
500 ml Perfusion Buffer
-
Buffer B
500ml HBSS
-
0.810 g (Final Conc 10 mM)
0.626g (5 mM)
-
2.5 g (5 mg/ml)
-
MgSO4
884 ul (Final Conc 1 mM)
-
240 µl (Final Conc 1.2
mM)
442 µl (Final Conc 1 mM)
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2. Begin by preparing laminin coated plates. Make up laminin (stored in 100 µg aliquots in the -20 C freezer)
solution in MEM media: Make up laminin at 10 µg/µl and coat the anticipated number of plates: 0.8 ml/well for a 6
well plate, 500 ul/well for 4 well chamber slide, 500 µl/well for a 12 well plate. Generally, the cells seem to attach
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best to 6 well plates coated with laminin. Incubate the coated plates at 37 C, 2% CO2.
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3. Prepare the apparatus. Turn on the water bath and warm water to approximately 42 C. Also turn on the water
pump. Perfuse the apparatus with sterile water (perfuse through a total of about 25 ml followed by flushing with
air). Then perfuse with 70% EtOH. Once the ethanol begins to elute, re-circulate and perfuse the apparatus for
about 15 minutes. Now flush the EtOH out with air and perfuse again with sterile H2O for a total of about 50 ml.
Once the water has passed completely through, perfuse with Perfusion Buffer. Make sure that the system is
completely devoid of air bubbles. This can be achieved by perfusing at a fast rate and while tipping the water
jacketed column and cannula upward and tapping the glassware.
4. Prepare the Enzyme Solution: For one heart, make 25 ml; For two hearts, make 30 ml; for three hearts, make 40
ml by adding the appropriate amount of perfusion buffer to all enzymes added to 1-50 ml falcon tube. Mix and
dissolve by vortexing and filter using a steriflip filter.
Enzyme /Amt
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Collagenase B 0.4 mg/ml (Stored 4 C)
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Collagenase D 0.3 mg/ml (Stored 4 C)
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Protease XIV 0.05 mg/ml (stored -20 C)
25 ml
30 ml
40 ml
10 mg
12 mg
16 mg
7 mg
1.25 mg
9 mg
1.5 mg
12 mg
2 mg
5. Prepare the serial, calcium gradient baths in 15 ml falcon tubes:
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Bath number
1
2
3
4
Buffer A
9.5 ml
8 ml
5 ml
0 ml
Buffer B
0.5 ml
2 ml
5 ml
10 ml
+2
Final [Ca ]
0.06 mM
0.24 mM
0.6 mM
1.2 mM
6. Make up the media. We generally use either plating or long term media. See the short term culture media (no
BDM) below as well.
MEM
Pen/Strep
L Glutamine
Stripped Bovine Growth
Serum
BSA (0.1 mg/ml)
ITS
BDM (10mM)
Plating Media
190 ml
2 ml
2 ml
10 ml
0.202 g
Short-Term Media
200 ml
2 ml
2 ml
-
Long-Term Media
200 ml
2 ml
2 ml
-
20.0 mg
-
20.0 mg
2 ml
0.202 g
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All media, once made, should be filtered and allowed to equilibrate for 2 hours in the 37 C / 2% CO2 incubator with
the caps loosened.
7. You will also need to prepare the cell filter which includes a bored out filter cassette with 250 µM nylon mesh
placed inside. This needs to be either autoclaved before hand or soaked in 70% EtOH for about 10 minutes, then
allowed to air dry and flushed with about 10 ml of sterile solution (perfusion buffer or buffer A).
8. Place surgical instruments in 70% EtOH and allow to soak for at least 5 minutes. Then remove and place in 100
2
cm dish (sterile) and allow to air dry.
9. Have ice cold sterile saline ready and pour approximately 7-8 ml into a sterile p60 culture dish. Keep on ice until
harvesting the heart.
Removal of the Heart:
1. Anesthetize the animal with pentobarbital (50 mg/ml diluted 1/10 in saline). Inject 0.25 ml intraperitonealy along
with 0.2 ml of heparin (1000 u/ml). Allow the drugs to take effect for about 10 minutes. If you’re doing two hearts,
you can anesthetize and give heparin to the other mouse now.
2. Tape the animal down onto the surgical tray, give an additional 0.1 ml of phenobarb if needed. Then prep the
chest by swabbing with 70% EtOH. Have the sterile saline (ice cold) ready on your right.
3. Prepare a pre-tied 5-0 silk suture to be ready to ligate the heart to the perfusion cannula.
4. Quickly open the chest. This is best done by first making a midline skin incision, from mid abdomen to the jaw,
then entering the peritoneum with the large scissors, clearing the diaphragm away by blunt dissection, and cutting
away the rib cage using the scissors with cuts up the chest wall on the lateral aspect of both sides. Snip away the
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fibrous connections between the heart and chest wall. Then cut away the rib cage all together. Using the small
forceps and scissors, gently lift the heart by the apex and expose the posterior aspect of the heart; cut away the
IVC, and veins and continue cutting from posterior to anterior until the heart is free. Place immediately in ice cold
saline.
5. Using the ultra fine forceps, identify the aortic root which emanates just posterior to the RV outflow tract. Grasp
the aortic wall and lift the heart. Using both forceps, open the lumen and place the aorta onto the perfusion
cannula. Then gently place the ligature around the aorta and tie as tightly as possible without moving anything. If
the aorta is too large to fit easily on the cannula, use the small Kelly clamps to fix the aorta to the cannula and then
tie the ligature. The best placement usually is with the aorta extending about 2 mm up on the cannula.
6. Begin perfusion at 3 ml/min. The heart should plump up nicely and appear as a homogeneous brownish pink
color. If you are satisfied with the appearance and the heart is secure, stop the perfusion briefly (1 sec) and place
the perfusion line into the enzyme media. Allow the about 10 ml of enzyme to perfuse and then re-circulate. At this
point, set the time for 7 minutes (total enzyme perfusion will therefore be about 10 min).
7. Clean up the instruments and working area and prep for the next animal.
8. Place 5 ml of buffer A into a sterile p60 cell culture dish.
9. After perfusion, remove the heart, place into buffer A in the p60, and gently cut away the atria and great vessels.
Gently pull the heart apart with the small forceps.
10. Using a sterile transfer pipette, aspirate the heart and cells up and down approximately 10 times to help
disperse the cells and tissue fragments. Then aspirate and pass through the nylon mesh filter (set on a 15 ml
falcon) that has already been rinsed or prepped with 5 ml of buffer A. After the solution has passed through, rinse
the filter with an additional 1 ml of buffer A and allow the cells to settle by gravity for 10-15 minutes.
11. While the cells are settling, you can hang the next heart after flushing the apparatus with sterile perfusion
buffer.
12. Next aspirate off the upper 13-14 ml of supernatant. If you want to culture fibroblasts, save the supernatant in a
50 ml falcon from as many hearts from of the same genotype as possible.
13. Aspirate the pellet gently with a sterile transfer pipette and place into bath 1. Again allow the cells to settle over
about 10 minutes. Repeat this for each subsequent bath. Once you reach bath 4, allow the cells to settle for
longer (15-20 minutes).
14. Aspirate off the supernatant. Resuspend the cells in plating buffer. Plate onto the appropriate number of
wells/plates after aspirating off the laminin. Plate for at least one hour, then change the media to long term media
detailed below. Alternatively, you could plate initially in the long term media and allow cells to attach overnight
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before changing the media again. All cells should be kept at 37 C with 2 % C02 atmosphere.
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