Labware

Soft-agar assay of using methylcelllose for HydroCell.

See photos on the web: suitable for screening of anti-cancer drug for NRK and A549 cells.

We add methylcellulose to increase viscosity of the culture meduim. Concentration as low as 0.6% methylcellulose would be easy for handling, stabilize medium and inhibit cells from making clusters by hitting each other while a float. Thereore, it allows highly precise measurement of cells, by alleviating necrosis inside the spheroid due to lack of nutrition and evading cell growth inhibition arouse from cell-cell contract and inhibit the adhesive growth.

1. Suspend cancer cells into 0.6% methylcellulose as quantiatively.
2. Seed cells onto HydroCell as many as 3,000 cells/cm² (We recommend cell density is under 10,000 cells/cm².)
3. Incubate for 3 days at CO2 incubator.
4. Take photo of cells, add the reagent for counting viable cells and measure OD after a few hours.

Protocol for preparation of methylcellulose

Materials: Methylcellulose 400cps (Wako, Cat#138-05052)

Procesure for 0.6% methylcellulose

Prepare 2.6% methylcellulose first, and then dilute into 0.6% methylcellulose.

A) Preparation of 2.6% methylcellulose

1. Add 2.6g of methylcellulose into 25mL of purified water (80~90 degree C) and dissolve completely by hard shaking.
2. Add 75mL of purified water (4 degree C) with hard shaking and stir with spinner cluster for more than 3 hours under 4 degree C until it becomes translucent.
3. Sterilize with autoclave at 121 degree C for 10 min, followed by cooling under 4 degree C for more than 24 hours until transparency is regained.

B) Preparation of 1.3% methylcellulose-medium mixture

Add equal medium (2 X MEM with serum) to 2.6% methylcellulose and stir for a few hours at room temperature before use.

Make 0.6% methylcellulose-medium mixture using B) prepared 1.3% methylcellulose- meduim mixture. Please use the medium adjusted for your cell types.