Spheroid-formation assay

Spheroid-formation assay

1. Seed cells in culture flask (Greiner Bio-One, Austria) as monolayer with mannosidase-inhibitor kifunensine (dissolved in DMSO, Sigma-Aldrich Chemie GmbH, Germany) in two different concentrations (1 and 10 µM) or DMSO in serum-reduced medium (5% FBS) and culture them for 72 h to reach a confluency of 80%. 
2. Coat each well of a 96-well plate (flat bottom) for cell culture with 50 µl of 2% agarose solution (SeaKem GTG Agarose, Lonza, Switzerland) in PBS for cell culture*.
3. Let the agarose dry and cool down under the bench for at least 3 hours. 
4. To generate cell spheroids seed 3 × 10³ pre-treated cells in 100 µl serum-reduced medium (5% FBS) with kifunensine or DMSO per well.
5. Do not use the edge rows of the 96-well plate for the experiment because of the risk of drying out (fill with liquid).
6. Every two days, add additional 20µl serum-reduced medium (5% FBS) with kifunensine or DMSO to each well by gently pipetting.
7. Check the growth of spheroids over 7 days and take pictures every 24 hours with light microscopy and camera (Axiovert 40 C, Carl Zeiss AG, Leica DFC320, Wetzlar, Germany).
8. The spheroid-formation ability of the cells was determined by comparing spheroid size after three different time points. 
    

* Solution preparation (For a 96-well plate):

1. Weigh 160 mg agarose into a 50 mL falcon and dissolve in 8 mL sterile PBS (Dulbecco’s Phosphate Buffer Saline without calcium and magnesium chloride) under the sterile bench.
2. Place falcon in a beaker with water (do not close the lid completely) and heat in the microwave: heat for 10 seconds, take out and shake carefully (HOT!!). Repeat this procedure until the agarose solution becomes clear!!!

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