- Humidified incubator (5% CO2 ± 1%, 36°C ± 2°C)
- Fluorescent micro plate reader
- 55 mM Iso-Osmolar tri-sodium citrate solution
Preparation of iso-osmolar tri-sodium citrate solution (traditional method)
- Prepare 55 mM tri-sodium citrate solution from 1M stock solution
- Adjust the osmolarity of citrate solution to that of the culture media using 100 g/L of NaCl solution. (Adding 1g/L NaCl to the solution will raise the Osmolarity by 30 mOsm)
- Adjust the pH with 1M citric acid solution to pH 7.2
Preparation of iso-osmolar tri-sodium citrate / glucose solution
- Dilute 55 mM Tri sodium citrate solution from 1M stock, and to that add 1 g/L of glucose
- Adjust the osmolarity using 100 g/L of NaCl solution (osmolarity of culture media)
- 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.
Spheroid generation on AlgiMatrix Plate
Plate the cells at a density of 10,000, 20,000, 40,000 and 80,000 cells/well in quadruplets 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. Culture the spheroids for 24 hours.
Isolation of spheroids by traditional method
- Aspirate the media from the well of AlgiMatrix plate using a 200 mL pipette.
- Add 200 µl of incomplete media to each well and incubate for 5 minutes at 37°C.
- Transfer the sponges to a 15 ml conical centrifuge tube.
- Add 1ml of 37°C iso-osmolar 55 mM tri sodium citrate solution per sponge
- Incubate the tube at room temperature for 4-5 minutes by gently mixing in a head to tail fashion.
- Spin the tube at 100 x g for 7 minutes at 25°C and discard the supernatant.
- Resuspend the spheroid pellet in complete medium and transfer to a 96 well black plate with clear bottom.
In situ harvesting of spheroids
- Aspirate the media from the wells.
- Add 250 µL of pre warmed 55 mM iso-osmolar tri-sodium citrate solution with 1gm/L glucose solution into each well and incubate for 10 minutes at 37°C.
- Spin the plate at 100 x g for 4 minutes at 25°C.
- Aspirate the tri-sodium citrate solution from the wells using a cut tip of mechanical pipette.
- (A).Repeat step 3 and spin the plate at 200 x g for 8 minutes at 25°C
(B). Aspirate the dissolved AlgiMatrix-citrate solution using cut tip and transfer it to 1.5 ml microfuge tube and spin the tube at 200 x g for 8 minutes.
- Collect and process the spheroids for down stream applications (DNA, RNA, Immuno Fluorescence and Protein Extraction)
CyQUANT GR staining
CyQUANT GR dye is a nucleic acid stain that can penetrate dead, but not live cell membranes. As dead cells have damaged membranes, the CyQUANT enters damaged cells and intercalates the nucleic acids. It produces fluorescence at ~520 nm when excited at ~480 nm.
- Add 200 µl of 1X CyQUANT GR dye in 1X DPBS in to each well containing spheroids
- Incubate the plate for 10 minutes at 37°C
- Read the plate at 480/520 nm (F min)
- Add 10 µl of 20X cell lysis buffer in to each well and mix it well
- Keep the plate at -70°C for 20 minutes
- Thaw the plate at room temperature and incubate for 10 minutes at 37°C
- Read the fluorescence at 480/520 (F max) (Consider as 100% dead cells)
Calculating the viability of spheroids
% of dead spheroids = F min / F max × 100
% of viable spheroids =100 – % of dead spheroids
F min= Fluorescence before cell lysis.
F max= Fluorescence after cell lysis.