Isolation of Eggs

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
    1. 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
    1. 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.