Methylcellulose medium is a type of semi-solid medium that can be used for culturing hematopoietic stem and progenitor cells in colony-forming assays, or culturing hybridomas and Chinese hamster ovarian cells for cell line development. This protocol provides instructions for preparing cultureware with semi-solid methylcellulose medium for downstream cell culture.

Materials

• Semi-solid methylcellulose medium (e.g. MethoCult™ or ClonaCell™)
• Blunt-end needle (e.g. Blunt-End Needles, 16 Gauge, Catalog #28110)
• Syringe (e.g. 3 cc Syringes, Catalog #28230)
• Culture dishes (e.g. 35 mm Culture Dishes, Catalog #27100)

Protocol

1. Semi-solid methylcellulose medium, such as MethoCult™ or ClonaCell™, must be thoroughly mixed prior to use to ensure a homogeneous mixture and best results for your assay. To mix, shake the bottle vigorously for at least one minute. You should see the medium become opaque with bubbles.
   
   
   Note: If required, add cytokines or other desired supplements first, and then shake thoroughly.
2. Wait 10 - 15 minutes for the bubbles to dissipate.
3. Aliquot the medium into working volumes using a blunt-end needle and syringe.
   
   
   Note: Do not use a pipette to aliquot methylcellulose medium. Methylcellulose medium is viscous and will stick to the inside of the pipette, causing the dispensed volume to be inaccurate.
4. Add the appropriate volume of concentrated single-cell suspension.
5. Mix by shaking vigorously for at least one minute.
   
   
   Note: For smaller aliquots, mix by vortexing for ~4 seconds.
6. Wait for the bubbles to dissipate.
7. Plate the sample using a blunt-end needle and syringe.