Qingqi Ren1, Chunlin Jiang2,3, Jikui Liu4. 1. Department of Hepatobiliary and Pancreatic Surgery, Peking University Shenzhen Hospital, Shenzhen, China. 2. Department of Medical Ultrasonics, Institute of Diagnostic and Interventional Ultrasound, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 3. Division of Interventional Ultrasound, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 4. Department of Hepatobiliary and Pancreatic Surgery, Peking University Shenzhen Hospital, Shenzhen, China liu8929@126.com.
Abstract
OBJECTIVE: To explore the application of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and MACSiBeads™ Particles within in vitro suppression assays of regulatory T (Treg) cells. METHODS: CD4+CD25+ Treg cells and CD8+ T cells were sorted using magnetically activated cell sorting. CD8+ T cells were subjected to CFDA-SE staining to determine their optimal staining concentration. MACSi-Beads™ Particles, a component of the Treg expansion kit, were used as stimulators in suppression assays. Five experimental groups were set based on the condition of MACSiBeads™ Particles, CFDA-SE staining and cell composition of co-culture system, which were stimulated CD8+ cells without CFDA-SE staining, unstimulated CD8+ cells with CFDA-SE staining, stimulated CD8+ cells with CFDA-SE staining, co-cultured with Treg cells at a ratio of 1:0.25, 1:0.125 and 1:0, respectively. Flow cytometry was performed using the BD FACS Canto™ and flow data was analyzed using FCS Express 4 Plus software. RESULTS: CFSE fluorescence intensity correlated with cell type and culture time. The final CFDA-SE staining concentration was 0.5μM. Within in vitro suppression assays, Treg cells showed a significant inhibitory effect on the proliferation of CD8+ T cells increasing as its concentration increased. CONCLUSION: CFDA-SE combined with MACSiBeads™ Particles can be used to evaluate the in vitro inhibition effect of Treg cells.
OBJECTIVE: To explore the application of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and MACSiBeads™ Particles within in vitro suppression assays of regulatory T (Treg) cells. METHODS:CD4+CD25+ Treg cells and CD8+ T cells were sorted using magnetically activated cell sorting. CD8+ T cells were subjected to CFDA-SE staining to determine their optimal staining concentration. MACSi-Beads™ Particles, a component of the Treg expansion kit, were used as stimulators in suppression assays. Five experimental groups were set based on the condition of MACSiBeads™ Particles, CFDA-SE staining and cell composition of co-culture system, which were stimulated CD8+ cells without CFDA-SE staining, unstimulated CD8+ cells with CFDA-SE staining, stimulated CD8+ cells with CFDA-SE staining, co-cultured with Treg cells at a ratio of 1:0.25, 1:0.125 and 1:0, respectively. Flow cytometry was performed using the BD FACS Canto™ and flow data was analyzed using FCS Express 4 Plus software. RESULTS: CFSE fluorescence intensity correlated with cell type and culture time. The final CFDA-SE staining concentration was 0.5μM. Within in vitro suppression assays, Treg cells showed a significant inhibitory effect on the proliferation of CD8+ T cells increasing as its concentration increased. CONCLUSION:CFDA-SE combined with MACSiBeads™ Particles can be used to evaluate the in vitro inhibition effect of Treg cells.