- 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.
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.