Holoclone Assay

Holoclone assay from Geroge/VLP

Prep

 * Filtered 15ug/mL mitomycin C (sigma M-0503) in serum-free DMEM (see protocol at bottom)
 * Phoenix cell medium (DMEM+10%FBS+pen/strep)
 * KGM
 * 3T3 cells ~ 50% or less confluent
 * Keratinocytes ready for plating
 * 6 cm plates (for now)

DAY 1

 * Aspirate phoenix cell media from 3T3 cells, wash 2x with serum-free DMEM
 * Add mitomycin+DMEM to cells; incubate for 2 hours at 37°C
 * Aspirate mitomycin+DMEM from cells, wash 2x with serum-free DMEM
 * Trypsinize 3T3 cells, spin, aspirate and resuspend in phoenix cell media
 * Count out 1 million to 1.5 million 3T3 cells, resuspend in phoenix cell media, plate out evenly on 6cm plate. Or if using 6 well plate= 400K-500k cells.
 * Let incubate overnight.

DAY 2

 * 1 hour before starting to plate keratinocytes, aspirate phoenix cell media and add 3mL KGM to 3T3 to precondition them
 * Prepare keratinocytes and resuspend in KGM, count to get 200 cells for a 6cm plate, dilute in 3mL KGM. For a 6 well plate use 80 cells.
 * Gently aspirate KGM from 3T3 cells that has been pre-conditioning; add back 3mL KGM + 200 keratinocytes
 * Place back at 37°C
 * Monitor cells and change KGM ( GENTLY ) every 3-4 days.
 * After 2 weeks, aspirate KGM and stain with Rhodamin B.

Making Mitomycin C stock and DMEM-resuspended solution

 * Start with 2mg mitomycin C powder (Sigma M-0503)
 * Add 2mL of molecular biology grade water with a syringe and needle through the top of the bottle.
 * Shake and resuspend until solution turns blue. This is your 1ug/ul master stock.
 * Add 15ul of master mitomycin C solution to each mL of serum-free DMEM for your drug solution.
 * Filter mito+DMEM through .45 micron filter.
 * Keep 15ug/mL mito+DMEM solution at 4°C, keep 1ug/ul master stock of mitomycin C wrapped in foil in -20°C