cd25 microbeads  (Miltenyi Biotec)

Journal: Journal of Clinical Apheresis

Article Title: Suppressive CD8 + T‐Cells Are Key Cellular Mediators of Extracorporeal Photopheresis

doi: 10.1002/jca.70094

Figure Lengend Snippet: Suppressive CD8+ T cells are increased in patients undergoing ECP. For all experiments PBMCs were isolated from whole blood obtained from healthy donors or patients undergoing ECP. (A) Flow cytometry was performed, and CD8+ suppressor cells were identified as indicated. (B–E) CD4+ CD25− cells were isolated from PBMCs, stained with CFSE, and incubated with CD8+ cells at the indicated ratios for 5–7 days. Flow cytometry was performed to identify CFSE dilute CD4+ cells (divided) and the % suppression was calculated by comparing the divided populations to CD4+ cells that were incubated in the absence of CD8+ cells (more details in the materials and methods). (B) CD8‐suppression assays in healthy donors versus ECP patients. (C) CD8‐suppression assays in paired samples obtained prior to the start of ECP (Baseline) or after 1 month of therapy. (D, E) Allo‐suppression assays. CD4+ CD25− cells were isolated from healthy donors or ECP patients. Each donor was paired with an ECP patient to allow for direct comparison between autologous and allogenic suppression results. (D) CD4+ CD25− cells from the healthy donors were incubated with CD8+ cells from the same healthy donor (auto) or a paired ECP patient (allo). (E) CD4+ CD25− cells from the ECP patients were incubated with CD8+ cells from a paired healthy donor (allo) or the same ECP patient (auto). Suppression of cell proliferation was evaluated as described above. Statistics were performed using Student's t ‐test with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Article Snippet: CD4+ CD25− cells were subsequently isolated from the CD8‐depleted cell population by negative selection using the CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐533) and CD25 Microbeads (Miltenyi Biotec, 130‐092‐983).

Techniques: Isolation, Flow Cytometry, Staining, Incubation, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Lactic acid improves Treg manufacturing and in vivo function

doi: 10.1016/j.omtm.2025.101600

Figure Lengend Snippet: Lactic acid enhances Treg expansion and purity Naive human Tregs (CD4 + CD45RO − CD45RA + CD127 − CD25 hi ) were stimulated using anti-CD3/CD28 Dynabeads and cultured for 9–15 days. At day 3, media was supplemented with 15 mM LA for the remainder of culture. (A) Schematic of Treg culture protocol. (B) Fold expansion and viability over time ( n = 18). (C) Expression of FOXP3 and Helios at day 9 ( n = 13). Representative figure shown on left. (D) Expression of CD25, CTLA-4, LAP, GARP, and CD39 at day 9 ( n = 10). Connected lines indicate individual donor pairs. (E and F) Treg media was supplemented with 15 mM LA, 15 mM sodium lactate (SL), or HCl (pH 6.7) at day 3. (E) Fold expansion and viability over time ( n = 9). (F) Expression of FOXP3 and Helios at day 9 ( n = 12). Statistical analysis was carried out by two-way ANOVA with Sidak’s multiple comparisons test (B and E), paired t test (C, left and D), Wilcoxon matched-pairs signed rank test (C, right), or Friedman test with Dunn’s multiple comparison’s test (F). Colored asterisks indicate significant differences between control and LA (blue), SL (green), or HCl (purple). Data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: To isolate Tregs from CD4 + T cells, CD25 + cells were separated using CD25 MicroBeads II (Miltenyi Biotec).

Techniques: Cell Culture, Expressing, Control