Experiments on the isolation and culture of chicken embryonic myoblasts
Experiments on the isolation and culture of chicken embryonic myoblasts
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Preparation of cell cultures
Materials and Instruments
HBSS Trypsin solution Collagen solution Move Equipment Preparation For more product details, please visit Aladdin Scientific website.
Dissecting microscope Stainless steel deep dish Dumom No. 5 forceps Lancet Fine spring scissors Tissue paper Large beakers Test tube racks Pipettes Hematocrit plates
1. Place the following items on the lab bench in the incubation room:
Dissecting microscope
Microscope bulb for surface and transmission illumination
Stainless steel deep tray with lid containing 70% alcohol
Dumom No. 5 pliers, minimum two pairs
Lancets 4
One pair of fine spring cutters
Wash bottle with 75% alcohol
Paper towels
Large beaker with discarded eggs
Paper towels for petri dishes
100 mm X 20 mm Petri dish
Covered stainless steel dish with Polytowel
Combination microscope with 100 magnification (objective X eyepiece) for cell counting
2. Place the following items on the ultra-clean table in the culture room:
Bunsen burner
A test tube rack that can be tilted to hold 15 ml test tubes.
Box containing: centrifuge tubes, Pasteur pipettes with cotton stoppers, pipette balls
Individually wrapped pipettes 1 ml, 5 ml and 10 ml
pumps
Blood counting plates
Hank's Balanced Salt Solution (HBSS) and Ca2+-freeMg2+-free HBSS
2.5% trypsin solution
2 Petri dishes of 35 mm size containing half the volume of HBSS
25 ml size flask
(1) Cut several sheets of lens paper into small rectangles 2 inches x 2.5 inches, cut a stack of filter paper into discs using the Swinny filter support plate as a template, and wash with ether in an ultra-clean bench for 2 hours, changing the ether solution 2 to 3 times.
(2) Wash with anhydrous ethanol for 2 hours, changing the solution 2 to 3 times.
(3) Wash the membrane with distilled water for 5-10 times and immerse it in water overnight.
(4) Take a coverslip, partially insert it into the water, drag a round piece of paper onto the coverslip with a small pair of pliers, and then place it face up on a paper towel on the ultra-cleaning table until the paper dries.
(5) Place the small round piece of paper mentioned above together with the coverslip in an airtight petri dish and store it, or carefully remove the small round piece of paper with small tongs and store it separately.
(6) Load the Swinney filter. Assemble the bottom half in the following order: gasket, membrane support, lens sheet, O-ring, and top half of the filter, loosely screwing the top and bottom parts together.
(7) Insert an 18# or larger gauge needle into the protruding outlet. Place in petri dish and autoclave in Fast-dry with autoclave tape.
(8) To use: tighten the filter and install the syringe advance lever.
3. Prepare collagen-coated tissue culture dishes in the ultraclean bench.
(1) Melt Collagen Solution (Life Technologies): overnight at 4°C or water bath at 37°C for 15 minutes.
(2) To make applicators: Melt the head of a Pasteur pipette over a gas flame and heat the next 1/2 inch until melted and bent at a right angle; allow to cool.
(3) Apply several drops of collagen solution to the plate. 1 to 2 drops are required for 35 mm size plates and 3 to 4 drops for 100 mm size plates.
(4) Use the applicator to spread the collagen evenly, making sure to cover the edges as well.
Collection and Dissection of Embryos
4. Wipe the laboratory table with 70% alcohol and place the dissecting mirror and lamp to one side.
5. lay out a paper towel on top of a row of petri dishes, one to hold the collected embryos and the rest to hold the opened eggs.
6. The beaker with the discarded eggs is also placed close by.
7. Carefully pinch the folded edge of the sterile towel out of the box and place it on the counting board with the folded edge facing away from you. Carefully open the towel by pinching the long edge of the towel. Take the instrument out of the alcohol, taking care that the tip is up, and then place it on the towel with the tip toward the folded edge and re-cover it.
8. Wipe or spray 11-day-old eggs with 70% alcohol, then crack and place in a petri dish. Hold the embryo gently by the neck with tongs and lift it out vertically into another petri dish. Collect all embryos in the same way. If the embryos are stillborn, throw them all away, including the petri dish.
9. The embryo is transferred to the dissecting microscope with light intensity to minimal surface light and the skin of the thorax is peeled off with forceps.
10. Holding the embryo in place with two forceps, the muscle is cut from the thoracic rib cage, a small cut is made close to and along the sternum, and then all the way down to the wing junction, the muscle contracts as soon as it leaves the point of attachment, and a small cut is made close to and along the thoracic rib cage and pulled down to the wing junction so that the muscle is completely detached, and then it is placed into a 35 mm Petri dish with HBSS solution, and the other half of the thoracic muscle is detached in the same manner. The other half of the pectoral muscle tissue was isolated in the same manner.
11. After all muscles were collected, they were examined under a dissecting microscope. The dissecting microscope is equipped with a more powerful transmitted light source than in step 9.
12. After cleaning all the muscles, place the petri dish on the dissecting microscope platform and use two scalpels to cut the muscle tissue into 1 mm3 pieces. Pasteurized pipettes with rubber bulbs are used to suck some HBSS solution into the petri dish.
13. The tissue blocks are picked up and transferred to a beaker containing 10 ml of 0.25% trypsin prepared with HBSS solution. The enzyme is free of calcium and magnesium ions. The beaker is wrapped in aluminum foil and incubated for 20-30 minutes at 37°C in a CO2 oven.
Trypsin digestion of cells
14. Transfer the muscle tissue to a centrifuge tube and centrifuge on a medical centrifuge at medium speed for 2 to 3 minutes so that the muscle tissue just settles to the bottom of the tube, but do not centrifuge it too long to form a solid mass.
15. Carefully pour the trypsin into a sterile petri dish.
16. Add 2 ml of muscle cell medium (8:1:1) to the centrifuge tube.
17. Check the tip of the cotton plugged Pasteur pipette to make sure that it is smooth and even without any cracks, and put on a suction ball to suck some medium to moisten its inner wall. Then insert it into the bottom of the centrifuge tube and blow gently.
18. 3 ml of 8:1:1 medium was added, blown slightly to disperse the cells, and then filtered through a Swinney filter with lens filter paper into a new tube.
Counting and Inoculating Cells
19. Count the cells.
20. Each 35 mm Petri dish is inoculated at a density of 3X105 cells/ml, for a total of 1.5 ml. For bulk cultures, e.g., 100 mm Petri dishes, 10 ml of 5X105 cells/ml per Petri dish is usually inoculated.
21. Incubate the cells at 37°C in a 5% CO2 water-jacketed incubator. For the first three days, change the medium daily, and thereafter, every other day.