Phosflow lyse fix buffer 5

Cells were pre-incubated with CD16/CD32 Fc block (BD PharMingen). Surface and intracellular cytokine staining was performed per manufacturer's instructions. PI or Ghost dye (Tonbo Biosciences, San Diego, CA, USA) was used to exclude dead cells and to measure cell viability where indicated. Phosflow staining was performed fixing the cells with BD Phosflow Lyse/Fix Buffer 5 × for 10 min at 37 °C, and then permeabilizing with BD Phosflow Perm Buffer III (BD Biosciences) for intracellular staining with Abs for phospho-proteins. Samples were acquired on a BD LSR II (BD Biosciences) and analysed using FlowJo software (Treestar, Inc., Ashland, OR, USA).

WEHI-231 cells and B220+CD23– cells from the BM (9-week-old mice) were incubated for 30 min in basal media alone or with the following signaling pathway inhibitors (10 μM): G129R to inhibit the PRL receptor, GSK690693 to inhibit PI3K (Sigma-Aldrich), and Stattic to inhibit STAT3. Subsequently, cells were incubated with PRL (50 ng/mL) for 30 min and fixed with 1× BD Phosflow Lyse/FIx Buffer 5× (BD Biosciences, San Jose, Cal, USA) for 10 min. Cells were permeabilized with Perm Buffer III from BD Phosflow (BD Biosciences) to determine STAT3 phosphorylation and activity in apoptosis rescue assays, and with IC Fixation Buffer (eBioscience) to determine AKT and ERK1/2 phosphorylation at 4 °C for 30 min. Cells were washed with FACS buffer or with Permawash and incubated at 4 °C for 30 min with the antibodies for flow cytometry analysis. Data were acquired using a MACSQuant Analyzer 10 cytometer (Miltenyi Biotec) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).