Plate and Mix Method

  1. Add gel to insert using positive displacement pipette
  2. Add media on top of gel and in external well
  3. Incubate for 20 minutes at 37 °C, 5 % CO2, humidified incubator
  4. Remove media from on top of gel carefully
  5. Remove media surrounding insert
  6. Repeat steps 2 – 5 twice (3 media changes over 1 hour)
  7. Alternatively, leave gel in media in the incubator between 1 hour and overnight
  8. Prepare cells (1-­‐4 million cells/mL, in 10uL of media per gel)
  9. Remove media from gels
  10. Pipette cells slowly into gel, with gently mixing, being careful not to introduce air bubbles
  11. Replace media around and on top of gels
  12. Culture as usual

 

Mix and Plate Method

 

  1. Prepare cells at 4million cells/mL in 200uL per 2mL of gel to use
  2. Aliquot maximum 2mL gel into an eppendorf, straight-­‐sided tubes are better than 1.5 mL pointy ones
  3. Gently and slowly mix 100uL cells into gel
  4. Repeat with remaining 100uL
  5. When satisfied that cells are homogeneously mixed use a positive displacement pipette to add gel + cells to insert
  6. Add media on top of gel and in external well
  7. Incubate for 20 minutes at 37 °C, 5 % CO2, humidified incubator
  8. Remove media from on top of gel carefully
  9. Remove media surrounding insert
  10. Repeat steps 4 – 7 twice (3 media changes over 1 hour)
  11. Culture as usual