Annals of clinical and laboratory science · 2019
CFDA-SE Combined with MACSiBeads™ Particles to Evaluate the Inhibitory Effect of Treg Cells in vitro.
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Ren Qingqi, Jiang Chunlin, Liu Jikui
Аннотация
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.
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.