Isolation of Viable Spheroids from Algimatrix™

Methods

1.    Spheroid isolation by using tri-sodium citrate

2.    Spheroid isolation by using Versene

Method by using Tri Sodium Citrate

  1. Aspirate the media from the well of Algimatrix™ plate
  2. Add 200 µl of incomplete media to each well and incubate for 5 minutes at 37°C
  3. Transfer 6 sponges to a 15 ml conical centrifuge tube
  4. Add 6 ml (1ml/sponge) of iso-osmolar 55mM tri-sodium citrate solution
  5. Incubate the tube at room temperature for 4-5 minutes by gently mixing in a head to tail fashion
  6. Spin the tube at 200xg for 7 minutes at 25°C and discard the supernatant
  7. Resuspend the spheroid pellet in complete medium for further use

Preparation of iso-osmolar tri-sodium citrate solution

  1. Prepare 55 mM tri sodium citrate solution from 1M stock solution
  2. 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)
  3. 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
MgCl2 has three
Tri-sodium citrate  has four (HOC(COONa)(CH2COONa)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).

Osmolarity of different media and salts

Media
Osmolarity in mOsms
Salts
Osmolarity in mOsms
DMEM
322-374
Earle’s BSS
271-308
RPMI
268-319
Hank’s BSS
265-305
Mc Coy’s 5A
275-320
Gly BSS
265-305
Ham’s F12
285-332
DPBS
280-311
MEM (Earl’s salts)
278-314
BME (Earls salts)
282-318
MEM (Hank’s salts)
274-317
(Hanks salts)
270-310

Method by using Versene

  1. Aspirate the media from the well of Algimatrix™ plate
  2. Add 200 µl of incomplete media to each well and incubate for 5 minutes at 37°C
  3. Transfer 5 sponges to a 15 ml conical centrifuge tube
  4. Add 10 ml Versene to the centrifuge tube containing 5 sponges, and place on an inverter at 37°C for 25 minutes
  5. Spin the tube at 200xg for 7 minutes at 25°C and remove supernatant
  6. Resuspend the spheroid pellet in complete medium for further use

Viability studies of Spheroids are done by using trypan blue dye exclusion method

  1. Mix equal volumes of spheroid suspension with 2 X trypan blue dye
  2. Place the mixture on a glass slide
  3. Observe the viability of spheroids under a phase contrast microscope

Reagents

1M Tri-sodium citrate 29.41 g/100 ml (Sigma Cat. #C2424)
1M Citric acid 19.213 g/100 ml (Sigma Cat. #S4641)
100 g/L sodium chloride (Sigma)
PBS (IVGN Cat. #20012-27)
Versene (IVGN Cat. #15040-066)
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