before start:

thaw cells from liquid nitrogen, grow in 75cc flask (T75) in Fischer's medium MM (maintenance medium) until content gets cloudy and starts to turn yellow (usually want ~ 6 x 10⁶ cells per T75), assay will be performed in 24-well-plate at concentration of 100,000 cells / well, each sample in triplicate.

One day before assay:

centrifuge cells, bring back to volume with Fischer's SM (stationary medium). It is important to remove all of MM from cells so that negative control is low. If wanted, cells can be rinsed in SM and spun again.

Day of assay:

spin cells, bring to smaller volume with SM and count, will want 100,000 cells / 0.5 mL / well; all proteins and controls done in triplicate. To wells containing 0.5 mL cell suspension add:

• 0.5 mL SM = negative control
• 0.5 mL MM = positive control
• oPRL = positive control (0.001 ng -10 ng (- 100 ng), in tenfold increments, diluted in SM to 0.5 mL total volume), max is reached at 1 - 10 ng
• sample proteins prepared in the same way (filter sterilize before using!)

incubate at 37°C for 72 hrs, then read on cell counter (Sysmex Microcellcounter CC 110)

Cell counter:

add 400 µL of test sustance to10 mL Hematall, count on counter. Reading of cell count x 57,000 is cell number in 1 mL original cell culture