Prior to start need:
- Petri dishes
- Autoclaved forceps or hemostat
- Autoclaved razor blades
- 70% ethanol (pure not junk)
- 500 ml of Dulbecco’s PBS (DPBS) with 10 ml of pen/strep/fungizone
- 0.5% collagenase (0.05 g of collagenase into 10 ml of dH20 (make fresh)
- cat # C5138-500mg
- Polymyxin B sulfate salt (cat # P4932-1 MU)
- Add 5 ml of sterile H20 to stock powder
- Incubate @37 C for 30 mins
- Aliquot into 500 ul in 1.5 ml tubes
- Prepare 3 petri dishes with 70% ethanol and wash for 1 min
- Prepare 3 petri dishes with DPBS (+Pen/strep) and wash
- Prepare 2 petri dishes (empty) for mincing of livers with razor blades until it is the consistency of pate.
- Change petri dishes (both DPBS and ethanol) every 5 livers processed
- Transfer minced livers to a 50 ml conical tube
- Add DPBS to 40 ml mark on tube
- Add 5 ml of 0.5% collagenase solution
- 200 ul of 100mg/ml Ampicillin
- Cap the tube and wrap the cap in parafilm
- Mix well
- Tape the tubes down to a tray
- Place the tray in 37 C incubator, with rocking setting overnight so that the tubes are rocking maximally
The next day
- Centrifuge the tube containing the digested livers at 400g for 5 mins
- Pour off the supernatant let the top bit of the pellet go with the liquid
- Resuspend the pellet in 50 ml 1X DPBS w/pen-strep-fungizone
- Repeat this wash 3 more times
- After the final wash, resuspend the pellets in 25 ml of DPBS
- Pass the suspension through the 250uM sieve in to a sterile beaker
- Pass the resulting filtrate through a second 150uM sieve
- Centrifuge the filtrate at 400g for 5 mins
- Add 3 mls pbs,
- Prepare a Percoll gradients mixing 8 ml Percoll with 32 ml of 0.25M sucrose (sterile filtered) in a 50ml corning tube, mix well
- 4.27 g of sucrose into a 50 ml tube with 50 ml of H20
- Centrifuge the gradient at 800 g for 10 min
- Remove all of the supernatent from the tube
- Wash the egg 3 times with DPBS
- Optional: repeat Percoll if eggs are still dirty with liver cells
- Make a second Percoll gradient in a 15 ml tube
- 7.5 ml sucrose and 2.5 ml Percoll and mix well
- Apply the resuspended eggs to the top of the new Percoll gradient and spin at 800g for 10 mins
- There will be very little trash on the top of the column. Remove the eggs with a pipette and put them in a fresh 15 ml tube
- Wash the eggs 3 times with DPBS
- On the last spin remove as much of the PBS as is humanly possible without getting eggs.
- Make a second Percoll gradient in a 15 ml tube