realease  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Microbiome-derived bile acid signatures in early life and their association with islet autoimmunity

doi: 10.1038/s41467-025-66619-6

Figure Lengend Snippet: a Schematic of the Th17 and iTreg differentiation protocol for primary human naïve CD4 + CD25 − T cells isolated from the umbilical cord blood of healthy neonates. CD4 + CD25 − T cells were activated with anti-CD3/anti-CD28 and differentiated into Th17 or iTreg cells in the presence of corresponding cytokines for 3 days. DMSO control or conjugated bile acids (Asn-UDCA, Ser-CDCA, Tyr-CDCA, and unconjugated UDCA) at 100 µM were added on day 0 of differentiation. Illustrations in a panel were created with BioRender.com (licensed). b – e IL-17a secretion in the supernatant of Th17 cultures treated with Asn-UDCA ( b ), Ser-CDCA ( c ), Tyr-CDCA ( d ), and UDCA ( e ) was quantified on day 3 of differentiation from four biological replicates using ELISA. f – i . Intracellular Foxp3 protein expression in iTregs cultured with Asn-UDCA ( f ), Ser-CDCA ( g ), Tyr-CDCA ( h ), or UDCA ( i ) was assessed on day 3 of differentiation by flow cytometry. Geometric mean fluorescence intensity (MFI) values are shown for six biological replicates. Statistical significance was determined using a paired, two-tailed Student’s t-test. Illustrations were Created in BioRender. Hirvonen, K. (2025) https://BioRender.com/5s3bnh7 . Asn–UDCA asparagine–ursodeoxycholic acid, Ser–CDCA serine–chenodeoxycholic acid, Tyr–CDCA tyrosine–chenodeoxycholic acid, UDCA ursodeoxycholic acid.

Article Snippet: CD4 + T cells were further enriched using CD4 + Dynal positive selection beads (Invitrogen, Cat# 11331D) followed by CD25 + T cell depletion using the CD25 Microbeads II kit (Miltenyi Biotec, Cat# 130-092-983), according to the manufacturer’s instructions.

Techniques: Isolation, Control, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Flow Cytometry, Fluorescence, Two Tailed Test