"Designing the Optimal Artificial Immune Synapse: Combining TCEs with Diakines" Image Files

Internalization Assay Human CD8⁺ T cells incubated with Dylight650-labeled DK210 (EGFR) along with FM™ 1-43 Dye (N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide). For conditions with neutralizing antibodies, anti-IL2Ra (R&D Systems) and anti-IL-10R1 (BioLegend) were incubated prior to DK210 (EGFR) addition. Synapse Imaging Human CD8+ T cells were cultured with Dylight650-labeled DK210 (EGFR) then cultured with A498-GFP and CD3xEGFR T-cell engager. Human CD8+ T cells were labeled with MemBrite580 (Biotium). TCR Co-localization Human CD8+ T cells were cultured with DylightTM 550-conjugated DK210 (EGFR) (100 ng/mL). T cells were stained with APC conjugated anti-CD3 prior to coculture with GFP Raji-B cells. Instrument: Images were taken using a Yokogawa CSU X-1 spinning disc confocal microscope.内化实验（Internalization Assay）：将人CD8⁺ T淋巴细胞与Dylight650标记的DK210（表皮生长因子受体EGFR）及FM™ 1-43染料（N-(3-三乙铵丙基)-4-(4-二丁氨基苯乙烯基)吡啶二溴盐）共同孵育。对于设置中和抗体的实验组，需预先加入抗IL2Ra抗体（R&D Systems公司）与抗IL-10R1抗体（BioLegend公司）进行孵育，随后再添加DK210（EGFR）。 突触成像（Synapse Imaging）：将人CD8⁺ T淋巴细胞与Dylight650标记的DK210（EGFR）共培养，随后与A498-GFP细胞及CD3xEGFR T细胞衔接蛋白共培养。使用MemBrite580（Biotium公司）对人CD8⁺ T淋巴细胞进行标记。 TCR共定位实验（TCR Co-localization）：将人CD8⁺ T淋巴细胞与Dylight™ 550偶联的DK210（表皮生长因子受体EGFR）（100 ng/mL）共培养。在与GFP标记的Raji-B细胞共培养前，使用别藻蓝蛋白（APC）偶联的抗CD3抗体对T细胞进行染色。 成像设备：采用Yokogawa CSU X-1型转盘式共聚焦显微镜采集图像。