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Spheroid isolation by using tri-sodium citrate
Preparation of iso-osmolar tri-sodium citrate solution
Calculating osmolarity in complex solutions:
As described under Units of Measure, the osmolarity of a simple solution is equal to the molarity times the number of particles per molecule.
Glucose has 1 particle
NaCl has two
MgCl 2 has three
Tri-sodium citrate has four (HOC(COONa)(CH 2COONa) 2)
Note: Osmolarity of 55 mm tri-sodium citrate solution is 220 mOsm and is adjusted to 340 mOsm if cells are cultured in DMEM (DMEM Osmolarity range 322-374).
Spheroid isolation by using Versene
Viability studies of Spheroids are done by using trypan blue dye exclusion method
- Aspirate the media from the well of Algimatrix™ plate
- Add 200 µl of incomplete media to each well and incubate for 5 minutes at 37°C
- Transfer 6 sponges to a 15 ml conical centrifuge tube
- Add 6 ml (1 ml/sponge) of iso-osmolar 55mM tri-sodium citrate solution
- Incubate the tube at room temperature for 4-5 minutes by gently mixing in a head to tail fashion
- Spin the tube at 200 x g for 7 minutes at 25°C and discard the supernatant
- Resuspend the spheroid pellet in complete medium for further use
Preparation of iso-osmolar tri-sodium citrate solution
- 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 1 g/L NaCl to the solution will raise the osmolarity by 30 mOsm)
- Adjust the pH with 1M citric acid solution to pH 7.2
Calculating osmolarity in complex solutions:
As described under Units of Measure, the osmolarity of a simple solution is equal to the molarity times the number of particles per molecule.
Glucose has 1 particle
NaCl has two
MgCl 2 has three
Tri-sodium citrate has four (HOC(COONa)(CH 2COONa) 2)
Note: Osmolarity of 55 mm tri-sodium citrate solution is 220 mOsm and is adjusted to 340 mOsm if cells are cultured in DMEM (DMEM Osmolarity range 322-374).
Media |
Osmolarity in mOsms |
Salts |
Osmolarity in mOsms |
DMEM |
322 - 374 |
Earle's BSS |
271 - 308 |
RPMI |
268 - 319 |
Hank's BSS |
265 - 305 |
McCoy's 5A |
275 - 320 |
Gly BSS |
265 - 305 |
Hanks F12 |
285 - 332 |
DPBS |
280 - 311 |
MEM (Earl's salts) | 278 - 314 |
BME (Earl's salts) |
282 - 318 |
MEM (Hank's salts) | 274 - 317 |
Hank's salts |
270 - 310 |
Spheroid isolation by using Versene
- Aspirate the media from the well of Algimatrix™ plate
- Add 200 µl of incomplete media to each well and incubate for 5 minutes at 37°C
- Transfer 5 sponges to a 15 ml conical centrifuge tube
- Add 10 ml Versene to the centrifuge tube containing 5 sponges, and place on an inverter at 37°C for 25 minutes
- Spin the tube at 200 x g for 7 minutes at 25°C and remove supernatant
- Resuspend the spheroid pellet in complete medium for further use
Viability studies of Spheroids are done by using trypan blue dye exclusion method
- Mix equal volumes of spheroid suspension with 2 X trypan blue dye
- Place the mixture on a glass slide
- Observe the viability of spheroids under a phase contrast microscope
LT120