Guava easycyte 6 l flow cytometer

Cryptotanshinone was acquired from Sigma (C5624–5MG). 5 nM Mb13 monobody was incubated with 20 µg/ml streptavidin-coupled Dynabeads M280 (Invitrogen) for 30 min on ice in BSS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8, 1 mg/ml BSA, 1 mM DTT, and 1 mM EDTA). The beads were then washed and blocked with 1 mM biotin for 30 min, followed by another wash and resuspension in BSS buffer. 200 nM SHP2 PTP was pre-incubated with or without 100 µM cryptotanshinone for 30 min at 30 °C in BSS buffer. Ten microliters of the Mb13-bead solution were then transferred to a well of a 96-well filter plate (MultiScreenHTS HV, 0.45 µm pore size; Millipore) and drained by vacuum. Twenty µl of the SHP2 PTP mixture were then transferred to the wells of the filter plate containing the drained beads and the plate was incubated at 30 °C with shaking for 30 min. The wells were drained and washed twice with ice-cold BSST (BSS buffer containing 0.1% Tween 20). After draining, 20 µl of 10 µg/ml of Dylight 650-conjugated to streptavidin (Thermo) in BSS was added to each of the wells. After incubation on ice with shaking for 30 min, the beads were washed with BSST twice in the same manner described as above. The beads were resuspended in 200 µl BSST and the fluorescence emission in the Red2 channel was analyzed for 5000 events on a Guava EasyCyte 6/L flow cytometer (Millipore).

The percentage of cells positive for phospho-Histone H3 (Ser10) were determined by flow cytometry. After ice-cold 70% ethanol fixation, cells were incubated in blocking solution (1% BSA in PBS) and with Alexa Fluor 488-conjugated with phospho-H3 (S10) antibody (Cell Signaling Technology) at a 1:100 dilution for 1 hour. Quantification of phospho-H3-positive cells were performed in a Guava Easycyte 6L flow cytometer (Millipore) and data was analyzed using FlowJo software (version 7.2.5) (FlowJo, OR, USA)