Spot Culture Protocol

Reagents and animals

Mice E13.5

Media for cultures
Total Volume = 4ml + (volume/well)*(# wells)

  • Neurobasal (Thermo 21103049 )
  • Pen/Strep 1:100
  • B27 Plus ((Thermo A3582801)  1:50
  • Glutamax (Thermo 35-050-061) 1:100
  • NGF (Sigma-Aldrich N6009) 1:1000
  • FDU 1:200- for neuron only culture

Dissection media and tools:

  • Fine forceps (2x #55, small scissors) –
  • Dissection Media; DMEM + Pen/Strep (1:100)

Dissociation media

.05% Trypsin-EDTA

Plates

  1. Coat plates for minimum 4 hours at 37ºC with Poly-D-Lysine (PDL; Thermo ICN10269490) 1mg/ml diluted 1:10 in sterile water.
  2. Wash 3x with water for 3 minutes each (can be stored at -20 ºC)
  3. Coat for 2-3 hours at 37oCwith Laminin 1:300 in sterile water
  4. Remove laminin and let dry completely
    • Leave lid off

Procedure

Dissections

  1. Open Uterus – grab by umbilical cord & transfer pups to new dish with dissection media.
  2. Holding ventral side, use #55 forceps to remove head and tail, cut down sides to empty viscera, hold down on either side and pull off any internal organs using #55
  3. Transfer back/spinal cord to new dish; make sure tissue is oriented ventral-side up. Use #55 forceps to spread tissue apart across spinal cord: separate with forceps between spinal cord and vertebrae.  Stick forceps under spinal cord and separate from the tissue, spinal cord should spiral away and release with the DRGs attached on the side. Hold the spinal cord, use #55 to remove DRGs

Spot culture

  1. Transfer the DRGs to an Eppendorf tube with dissection media using p1000
  2. Centrifuge at 500 x g for 2 min and pipette off media
  3. Add 700ul .05% Trypsin-EDTA and incubate at 37oC for 25 min
    • After 10-15 minutes invert the tube 5 times and reincubate
  4. Centrifuge at 500 x g for 2 min, remove supernatant, add 700ul culturing media, and triturate 25x with p1000
    • Repeat 3x total
  5. Resuspend cells in enough culturing media for 2.5ul/spot + 10% extra
  6. Spot 2.5ul in the wells of each plate
  7. Wait ~10 minutes
    • Watch to make sure the spots don’t dry up
  8. Slowly add culturing media to the edge of the wells