Coated no 1.5 coverslips

Immunostaining was performed as described previously ^{36 (link)}. In brief, U2OS or TZM-bl cells were grown on poly-L-lysine (Sigma-Aldrich)-coated No. 1.5 coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 minutes, and then blocked with 5% bovine serum albumin in phosphate-buffered saline. The cells were stained with the indicated primary antibodies and appropriate Alexa fluorophore–conjugated secondary antibodies (Thermo Fischer Scientific) listed in Supplementary Table 3. The resulting samples were mounted with ProLong Gold Antifade (Thermo Fisher Scientific) before microscopic analysis.
To prepare thin-layered samples with suspension cultures (CEM-SS and Human CD4⁺ T cells), 0.25 × 10⁶ cells (400 μL) were put into a cuvette assembled with a poly-L-lysine (Millipore Sigma)-coated No. 1.5 coverslip (Marienfeld Superior) and a cytoclip slide clip, then centrifuged at 1,500 rpm for 4 minutes using Cytospin 3 (Thermo Scientific Shandon). The resulting cells mounted on coverslips were fixed with 4% paraformaldehyde or 1:1 methanol and acetone mixture (for γ-tubulin staining only) and then immunostained as described above.

Immunostaining was performed as described previously^{36 (link)}. In brief, U2OS or TZM-bl cells were grown on poly-L-lysine (Sigma-Aldrich)-coated No. 1.5 coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min, and then blocked with 5% bovine serum albumin in phosphate-buffered saline. The cells were stained with the indicated primary antibodies and appropriate Alexa fluorophore–conjugated secondary antibodies (Thermo Fischer Scientific) listed in Supplementary Table 3. The resulting samples were mounted with ProLong Gold Antifade (Thermo Fisher Scientific) before microscopic analysis.
To prepare thin-layered samples with suspension cultures (CEM-SS and Human CD4⁺ T cells), 0.25 × 10⁶ cells (400 μL) were put into a cuvette assembled with a poly-L-lysine (Millipore Sigma)-coated No. 1.5 coverslip (Marienfeld Superior) and a cytoclip slide clip, then centrifuged at 300 × g for 4 min using Cytospin 3 (Thermo Scientific Shandon). The resulting cells mounted on coverslips were fixed with 4% paraformaldehyde or 1:1 methanol and acetone mixture (for γ-tubulin staining only) and then immunostained as described above.