One cellstar

2500 cells per well were plated in 12-well plates (Greiner Bio One Cellstar, Frickenhauser - Germany) and were allowed to grow for about 4 to 5 days until small colonies could be clearly seen. Cells were treated for 48 hrs with different concentrations (2–100 nM) of staurosporine or UCN-01 (7-hydroxystaurosporine) in growth media. For each concentration datapoint of the two drugs, cells were analyzed in quadruplicates. Staurosporine was purchased as 1 mM ready-made solution in DMSO (Sigma Cat # S6942) and UCN-01 as powder (Sigma Cat # U6508). UCN-01 was diluted in DMSO according to the manufacturer's instructions. Cell culture plates containing colonies were gently washed with PBS and fixed with 3.7% formaldehyde for 10 minutes. Wells were rinsed once again with PBS and colonies were stained with 0.2% crystal violet solution in 10% ethanol for 10 minutes. Excess stain was removed by washing repeatedly with PBS. All the procedures were done at room temperature. The plates can be stored at room temperature or at +4 °C for several months without any visible fading of the dye.

Relative Cdc42 activities were measured using a colorimetric G-LISA assay (Cytoskeleton, Denver, CO). RBL-2H3 and B6A4C1 cells were plated in 35 mm dishes (Greiner Bio One Cellstar) at a density of 1×10⁵ cells/ml and sensitized with IgE. Cells were then washed once with buffered saline solution (BSS: 135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂ 5.6 mM D(+) glucose, 20 mM HEPES, pH 7.4) and cells were stimulated for either one or three minutes at 37°C with 0.2 µg/ml multivalent DNP-BSA (Posner et al., 1992 (link)), then processed according to the manufacturer's instructions, except that a lysis buffer containing 25 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% (v/v) Triton 100, 1 mM sodium orthovanadate, 1 mM β-glycerol phosphate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin was used.

For β-gal assays, an over-night culture was used to inoculate (1%) 50-mL LB flasks that were incubated at either 25 or 37°C with 200 rpm shaking. For the assay, at each time point, 2 × 200 μL of each culture were collected into a sterile 96-well plate (F-bottom) (Greiner Bio-One Cellstar) and then frozen at −80°C. Using a 96-well microplate reader SPECTROstarNano (BMG Labtech) and the MARS Data Analysis software (BMG Labtech), the rate of β-gal production was measured at OD₄₂₀ following the methods of Schaefer et al. (61 (link), 62 (link)) with a custom β-gal mix: 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl,1mM MgSO₄; 1.8 μL mL⁻¹ β-mercaptoethanol, 0.2 mg mL⁻¹ Lysozyme from chicken egg (Sigma), 1:150 diluted Bacterial Protein Extraction Reagent (Thermo Fisher); and 1 mg mL⁻¹ of 2-nitrophenyl-β-galactopyranoside (Sigma). To control for temperature-dependent differences in the MH96 calibration curves measured at OD₆₀₀ for 25 and 37°C, an appropriate calibrating factor was applied to the Miller Unit Equivalent calculation.