Journal: STAR Protocols

Article Title: Protocol for the preparation and analysis of patient-derived organotypic tumor spheroids

doi: 10.1016/j.xpro.2025.104286

Figure Lengend Snippet: PDOTS surface marker staining (A) Scheme showing the steps for surface marker staining of PDOTS “on chip”. (B) Surface markers staining of melanoma PDOTS. The left image is an overlay of the indicated markers. White arrows denote CD38 + CD8 + T cells inside a tumor spheroid.

Article Snippet: CD8 MicroBeads , Miltenyi , Cat#130-045-201.

Techniques: Marker, Staining

Journal: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Tregs traffic from the tumor to the TdLN at higher proportions and are equally susceptible to radiation as non-Treg CD4 T cells (A) Kaede mice were injected with either MC38, MOC1, or MOC2 cancer cells subcutaneously. Tumors were photoconverted on day 14 via ultraviolet light. TdLNs were harvested on day 3 post-photoconversion. All cell populations were pre-gated on live singlets. (B) Photoconverted CD8 T cells were defined as CD3 + CD4 − CD8 + Red + . Photoconverted Tregs were defined as CD3 + CD8 − CD4 + CD25 + Red + . Photoconverted non-Treg CD4 T cells were defined as CD3 + CD8 − CD4 + CD25 − Red + . (C) Frequencies of photoconverted CD8 T cells, Tregs, and non-Treg CD4 T cells were quantified via flow cytometry. (D) Mice were injected with MC38 cancer cells subcutaneously. Tumors were treated with 12 Gy RT on day 14 and harvested for analysis via flow cytometry on day 1–3 post-RT. Non-Treg CD4 T cells and Tregs frequencies as a percentage of live cells were quantified. (E) MC38-bearing Kaede mouse tumors were photoconverted on day 14 followed by immediate treatment with 12 Gy RT. (F) TdLNs were harvested on day 1–3 and percentages of photoconverted non-Treg CD4 T cells and Tregs were analyzed via flow cytometry. A total of 5–6 mice were analyzed per group. Data are represented as mean ± SD. Statistics were performed using an unpaired Student’s t test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: CD4/CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat #: 130-116-480.

Techniques: Injection, Flow Cytometry

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: The protective effect of ECP is dependent on CD8+ T cells. For all experiments 6–8 week old female mice of the indicated strain were used. EAE was induced with the indicated peptide on Day 0 and mice were monitored daily for signs of disease. (A) ECP was performed using spleen + LN cells obtained from PLP 139‐151 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. N = 17 mice/group. (B) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient CD8 −/− mice on Day 0. Control mice received no cells. N = 11 mice/group. (C, D) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. On Day 14 of EAE, mice were euthanized, spleens and LNs were harvested, and cells were incubated with IL‐2 and PLP 178‐191 for 72 h. CD8+ T‐cells were isolated, and five million cells were transferred to recipients at the time of EAE induction with PLP 178‐191 . Control mice received no cells (control), the remainder of the mice received CD8+ T cells obtained from ECP treated (cells from ECP‐treated) or untreated (cells from control) mice. N = 6 mice/group. Statistics were performed using Student's t ‐test with *** indicating p < 0.001.

Article Snippet: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

Techniques: Control, Incubation, Isolation

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: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

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