CD4+ T-cell Proliferation Assay

The 96-well plates were precoated with 10 µg/mL anti-mouse CD3 mAb (BioLegend) and 5 µg/mL anti-mouse CD28 mAb (BioLegend) to stimulate CD4⁺T-cells. The total CD4⁺T-cells were isolated from spleens of naive mice by positive CD4⁺T-cell isolation kit (Miltenyi Biotec, USA). The cells were then labeled with 1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) for 10 min and co-cultured with MSC-sEVs at a concentration of 10 µg/ml. After 72 h of incubation, the CFSE fluorescence intensity was measured by FACS and analyzed by FlowJo.

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Duan Y., Chen X., Shao H., Li Y., Zhang Z., Li H., Zhao C., Xiao H., Wang J, & Zhang X. (2024). Enhanced immunosuppressive capability of mesenchymal stem cell-derived small extracellular vesicles with high expression of CD73 in experimental autoimmune uveitis. Stem Cell Research & Therapy, 15, 149.

Publication 2024

anti-mouse CD3 mAb anti-mouse CD28 mAb positive CD4+T-cell isolation kit carboxyfluorescein diacetate succinimidyl ester (CFSE)

Corresponding Organization :

Other organizations : Tianjin Medical University, Tianjin Medical University Eye Hospital, University of Louisville

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Variable analysis

independent variables

Concentration of anti-mouse CD3 mAb (10 µg/mL)
Concentration of anti-mouse CD28 mAb (5 µg/mL)
Concentration of MSC-sEVs (10 µg/ml)

dependent variables

CFSE fluorescence intensity (measured by FACS)

control variables

Isolation of total CD4+ T-cells from spleens of naive mice
Labeling of CD4+ T-cells with 1 µM CFSE for 10 min
Incubation time (72 h)

positive control

Not explicitly mentioned

negative control

Not explicitly mentioned

Annotations

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