Acetylated tubulin immunofluorescence

Β16F10 (4 × 10⁴) cells pretreated with 50 nM ZMP or carrier control (DMSO 1:2000 dilution) for 3 h were trypsinized and seeded into a μ-Slide VI0.4 ibiTreat (ibidi) coated with fibronectin (5 µg/ml at 37°C for 45 min). Human Effector T cells (at a density of 2 × 10⁶/ml) pretreated with 100 nM ZMP (a nontoxic dose found optimal for MT stabilization) or carrier control (DMSO 1:1000 dilution) for 6 h were washed twice with warm PBS, settled into a μ-Slide VI0.4 ibiTreat (ibidi) precoated with 100 ng/ml CXCL12 (PeproTech, Cat# 300-28A) overnight, and 0.01% poly-L-lysine (Sigma-Aldrich, Cat# P4707) for 1 h at room temperature (RT). After 30 min, cells were fixed with paraformaldehyde (4% in PBS) for 15 min at RT and permeabilized with a solution of 0.25% Triton X-100 for 15 min at RT. Cells were then incubated with anti-acetylated α-tubulin antibody (Santa Cruz Biotechnology Cat# sc-23950, RRID:AB_628409) for 1 h at RT in a solution of 0.025% Triton X-100 and 1% BSA in PBS. Cells were washed three times with PBS and labeled with a Alexa Fluor488-conjugated secondary antibody (Thermo Fisher Scientific, Cat# A-21202, RRID:AB_141607), for 1 h at RT, and further labeled with 20 µM Hoechst 33342 for 5 min at RT. Cells were imaged using an IX83 inverted microscope (described above) equipped with a PLAPON 60×OPh/1.4 objective (Olympus) using a Chroma filter set.