Journal: STAR Protocols

Article Title: Protocol for co-culture of murine bone marrow-derived dendritic cells with OT-I mouse CD8 + T cells to evaluate immune activation efficacy

doi: 10.1016/j.xpro.2026.104621

Figure Lengend Snippet: Purity of CD8 + T cells after magnetic isolation Representative flow cytometry analysis showing the percentage of CD8 + T cells following negative selection.

Article Snippet: CD8a FITC clone 53–6.7 (Dilution: 2 μL in 100 μL FACS buffer) , Miltenyi Biotec , Cat. no. 130-118-468.

Techniques: Isolation, Flow Cytometry, Selection

Journal: STAR Protocols

Article Title: Protocol for co-culture of murine bone marrow-derived dendritic cells with OT-I mouse CD8 + T cells to evaluate immune activation efficacy

doi: 10.1016/j.xpro.2026.104621

Figure Lengend Snippet: Antigen-specific CD8 + T cell activation after co-culture with transfected BMDCs (A) Percentages of IFN- γ -producing CD8 + T cells after 6 h of co-culture with transfected BMDCs. (B) CD8 + T cell proliferation assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution after 72 h of co-culture, shown as the percentage of divided cell subsets.

Article Snippet: CD8a FITC clone 53–6.7 (Dilution: 2 μL in 100 μL FACS buffer) , Miltenyi Biotec , Cat. no. 130-118-468.

Techniques: Activation Assay, Co-Culture Assay, Transfection

Journal: iScience

Article Title: Targeting KIF20A blocks lactylation modification to suppress immune escape in hepatocellular carcinoma

doi: 10.1016/j.isci.2026.115372

Figure Lengend Snippet: Glycolysis inhibition-mediated blockade of H3K18la in HCC cells restores the function of co-cultured CD8 + T cells (A) Schematic diagram of the experimental design. (B) Changes in lactate levels in Huh7 cells after treatment with 2-DG or oxamate. (C–E) Representative WB results showing the levels of Pan Kla and H3K18la in Huh7 cells treated with inhibitors. (F) Lactate levels in DKO cells with or without lactate supplementation. (G and H) WB analysis of the protein levels of LDHA, LDHB, Pan Kla, and H3K18la in DKO cells with or without lactate treatment. H3 and β-actin were used as loading controls for lactylation markers and LDH isoforms, respectively. (I) Colony formation assays show the proliferation of HCC cells (control group vs. DKO group) cultured alone or co-cultured with CD8 + T cells. (J) Quantitative analysis of the proliferation of co-cultured CD8 + T cells by FC. (K) FC analysis of the expression levels of GzMB (L) and IFN-γ (M) in co-cultured CD8 + T cells. All data are shown as mean ± SD. (B, D, E, and G) were analyzed using one-way ANOVA, (J) analyzed by two-way ANOVA, and (K) was analyzed by Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Experiments were repeated three times.

Article Snippet: APC Anti-Mouse CD8a Antibody , Elabscience , Cat# E-AB-F1104E; RRID: AB_3740201.

Techniques: Inhibition, Cell Culture, Control, Expressing

Journal: iScience

Article Title: Targeting KIF20A blocks lactylation modification to suppress immune escape in hepatocellular carcinoma

doi: 10.1016/j.isci.2026.115372

Figure Lengend Snippet: Knockdown of LDHA/B inhibits HCC growth by activating CD8 + T cell-mediated antitumor immunity (A) WB analysis of LDHA, LDHB, and β-actin (loading control) expression in control and LDHA/B DKO Hepa 1–6 cells. (B) Representative images of tumors dissected from tumor-bearing mice ( n = 6). Tumor weights (C), tumor growth curves (D), Kaplan-Meier survival curve ( n = 6) (E), and lactate contents in tumor tissues (F) of the control and LDHA/B DKO groups ( n = 6). (G) IHC staining of H3K18la and Pan Kla in tumor tissues (scale bars, 100 μm, n = 6). (H–M) FC analysis of tumor-infiltrating immune cells: TILs were isolated from control and LDHA/B-DKO tumors. FC plots (left) and quantitative statistics (right) show: the proportion of CD3 + CD8 + CTLs (H), the proportion of CD44hiCD62Llo effector memory CTLs (I), and the proportions of intratumoral GzMB + (J), and IFN-γ + (K) CTLs with representative scatterplots ( n = 6). Cell aggregates and debris were first excluded, followed by gating on the target cell populations and markers of interest. Representative FC plots and quantitative data are presented. (L and M) Tumor size and growth curves of mice treated with anti-CD8 antibody to deplete CD8 + T cells or isotype control (IgG) ( n = 6). (N) Mouse body weights at the end of the experiment ( n = 6). Data are presented as mean ± SD. (C–F, and H–M) were analyzed using Student’s t test, and (N) was analyzed by two-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, not significant. All experiments were repeated three times. β-actin served as the loading control for WB experiments.

Article Snippet: APC Anti-Mouse CD8a Antibody , Elabscience , Cat# E-AB-F1104E; RRID: AB_3740201.

Techniques: Knockdown, Control, Expressing, Immunohistochemistry, Isolation

Journal: iScience

Article Title: Targeting KIF20A blocks lactylation modification to suppress immune escape in hepatocellular carcinoma

doi: 10.1016/j.isci.2026.115372

Figure Lengend Snippet: KIF20A regulates PD-L1 expression via c-Myc (A and B) Huh7 and HCCLM3 cells were transfected with KIF20A small interfering RNAs (siRNAs) or KIF20A overexpression (OE) pcDNA3.1 plasmids, respectively. The protein levels of KIF20A, c-Myc, and PD-L1 were measured by WB in Huh7 and HCCLM3 cells. (C) ChIP-PCR was performed to assess the binding of c-Myc to the PD-L1 promoter region in Huh7 cells. (D) Huh7 cells were transfected with c-Myc siRNAs, and the knockdown efficiency of c-Myc was verified by RT-PCR. (E and F) Huh7 and HCCLM3 cells were co-transfected with KIF20A OE plasmids and c-Myc siRNAs. The protein levels of KIF20A, PD-L1, and c-Myc were determined by WB. (G) Huh7 cells were co-transfected with pGL3-PD-L1 or pGL3-basic plasmids, KIF20A OE pcDNA3.1 plasmids, c-Myc siRNAs, and pRL-TK plasmids, followed by luciferase activity assay. (H and I) KIF20A promotes HCC cell immune evasion by regulating PD-L1 expression via c-Myc. HCC cells from different groups were co-cultured with CD8 + T cells. Representative FC plots and quantitative analysis of GzMB and IFN-γ expression in CD8 + T cells co-cultured with Huh7 cells. (J and K) Representative images and quantitative analysis of colony formation in Huh7 cells with or without CD8 + T cell co-culture. Data are presented as mean ± SD. Statistical analyses were performed using Student’s t test (D), one-way ANOVA (C, I), or two-way ANOVA (G, K). ∗∗ p < 0.01 and ∗∗∗ p < 0.001. All experiments were repeated three times.

Article Snippet: APC Anti-Mouse CD8a Antibody , Elabscience , Cat# E-AB-F1104E; RRID: AB_3740201.

Techniques: Expressing, Transfection, Over Expression, Binding Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Cell Culture, Co-Culture Assay