Spheroid generation on AlgiMatrix™ plates

Plate the cells at a density of 20,000 - 80,000 cells/well in triplicates in 100 ml complete media on AlgiMatrix™ plate.   Spin the plate at 100xg for 4 minutes.  Incubate the plate at 37°C in a 5% CO2 incubator for 10 minutes.  Add 130 ml of complete media subsequently into each well.

In Situ harvesting of spheroids by tri-sodium citrate method

  1. Aspirate the media from the wells
  2. Prepare 55 mM iso-osmolar tri-sodium citrate solution with 1g/L glucose and bring the solution to 37°C
  3. Add 250uL of pre-warmed tri-sodium citrate solution into each well and incubate for 10 minutes at 37°C
  4. Spin the plate at 200xg for 4 minutes at 25°C
  5. Aspirate the tri-sodium citrate solution from the wells with a mechanical pipette and a cut tip
  6. Repeat steps 3, 4 and 5
  7. Collect and process the spheroids for down stream applications (DNA, RNA, immunofluorescence and protein extraction)

Preparation of iso-osmolar tri-sodium citrate/glucose solution

  1. Dilute 55 mM tri-sodium citrate solution from 1M stock solution, to that add 1 g/L glucose
  2. Adjust the osmolarity using 100 g/L of NaCl solution (osmolarity of culture media)
  3. Adjust the pH with 1M citric acid solution to pH 7.2 - 7.4

Note:  Adding 1g/L NaCl to the solution will raise the osmolarity by 30 mOsm

Materials

Algimatrix Plate (IVGN Cat. #12684-015)
PBS 1X (IVGN Cat. #14190-300)
Tri-sodium citrate (Sigma Cat. #C2424)
Citric acid (Sigma Cat. #S4641)
NaCl (Sigma)
Alamar Blue (IVGN Cat. #DAL1025)
Humidified incubator (5% CO2 ± 1%, 36°C ± 2°C)