Goat anti mouse igg2a alexa fluor 568

A 22 × 22 mm cover glass was placed on each well of a 6‐well plate. Anti‐CD3 (BioLegend, 100340, 10 µg mL⁻¹) and anti‐CD28 (BioLegend, 102116, 2 µg mL⁻¹) diluted in PBS coated each well and were incubated at 4 °C for 12 h. After suctioning the coating solution, isolated CD4⁺ or CD8⁺ T cells in complete RPMI supplemented with sodium pyruvate, L‐glutamine, and 2‐mercaptoethanol were placed in each well. T cells were treated with 50 µg mL⁻¹ ICAM‐1 and incubated at 37 °C for 1–2 h in a 5% CO₂ atmosphere. The secondary antibody was treated at 1:1000 dilution. Cells were observed using a confocal laser scanning microscope (Carl Zeiss, LSM 800) and analyzed using Zen 2.3 (blue edition, Carl Zeiss Microscopy GmbH, 2011). The secondary antibody information was as follows: Goat anti‐human IgG Alexa Fluor 568 (Thermo Fisher Scientific, A‐21090, RRID:AB_2535746), goat anti‐mouse IgG2a Alexa Fluor 568 (Thermo Fisher Scientific, A‐21134), DAPI solution (Abcam, ab228549), and phalloidin‐Fluor 488 reagent (Abcam, ab176753).