L alexa fluor 555

Bone marrow derived macrophages (BMDMs) were collected from tibia of WT or KΔ75 mice. Isolated bone marrow cells were cultured in RPMI, supplemented with 10% heat-inactivated FBS, penicillin-streptomycin, GlutaMax and pyruvate in a 10-mm petri dish. Cells were differentiated with 10% L-cell conditioned medium (LCCM) for 10 days. On day 10, BMDMs were plated into non-tissue culture treated 6-well plates without LCCM overnight. The next day, BMDMs were collected for stimulation with 20ng/ml IL-10 for 15 min at 37 °C. At the end of the stimulation, cells were fixed in 2% paraformaldehyde for 15 min at 22 °C, followed by permeabilization with ice-cold 90% methanol on ice for 30 min. Cells were then stained with primary antibodies in 0.5% BSA PBS for 1h at 22 °C. Primary antibodies used were pSTAT3 (1:100, D3A7, rabbit, Cell Signaling Technology, #9145) and STAT3 (1:100, 124H6, mouse, Cell Signaling Technology, #9139). After washing with PBS twice, cells were stained in secondary antibodies: anti-rabbit IgG Alexa Fluor 555 (donkey, 1:1,000; Abcam, ab150074) and anti-mouse IgG(H+L) Alexa Fluor 555 (goat, 1:1,000, Invitrogen, A21424). Samples were acquired on a BD FACSCalibur Flow Cytometer and data were analyzed using FlowJo (10.8.1).