ldh level in ac16 cells measured using promega cytotox 96® kit  (Promega)

Journal: Oncology Letters

Article Title: Lung squamous cell carcinoma downregulates haptoglobin expression to inhibit M2 macrophage ferroptosis via the hemoglobin-dependent CD163/HO-1 pathway

doi: 10.3892/ol.2026.15558

Figure Lengend Snippet: Overexpression of HP in LUSC cells does not induce ferroptosis in LUSC cells. (A and B) NCI-H226 and NCI-H520 cells were infected with LV- HP or LV-Ctrl. After 72 h of infection, cells and culture supernatants were collected for analysis of HP (A) mRNA and (B) protein levels by RT-PCR and ELISA, respectively. (C) NCI-H226 and NCI-H520 cells infected with LV- HP or LV-Ctrl were cultured with or without Hb (30 µg/ml) for 24 h. Culture supernatants were collected and LDH release was measured. (D) Cells were analyzed via flow cytometry to determine the percentage of 7-AAD-positive cells in NCI-H226 and NCI-H520 cells. (E and F) The mean fluorescence intensity of (E) FerroOrange (Fe 2+ ) and (F) C11-BODIPY (lipid peroxide) was measured using flow cytometry. Data are presented as the mean ± SD from five independent experiments and were analyzed using an unpaired t-test or a two-way ANOVA followed by a Tukey's post-hoc multiple comparison analysis. ****P<0.0001. ns, not significant; HP, haptoglobin; Hb, hemoglobin; LDH, lactate dehydrogenase; 7-AAD, 7-aminoactinomycin D.

Article Snippet: An LDH cytotoxicity assay kit (cat. no. HY-K1090; MedChemExpress) was used to assess LDH levels in the supernatant of LUSC cell lines (NCI-H226 and NCI-H520 cells) and M2 macrophages, according to the manufacturer's protocol.

Techniques: Over Expression, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Fluorescence, Comparison

Journal: Oncology Letters

Article Title: Lung squamous cell carcinoma downregulates haptoglobin expression to inhibit M2 macrophage ferroptosis via the hemoglobin-dependent CD163/HO-1 pathway

doi: 10.3892/ol.2026.15558

Figure Lengend Snippet: CM from LUSC cells overexpressing HP leads to ferroptosis in M2 macrophages. (A) THP-1 cells were differentiated into macrophages by treating them with 150 nM PMA for 24 h, followed by culture in fresh RPMI-1640 for 24 h. The macrophages were polarized into M2 macrophages by incubating with IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 24 h. Flow cytometry was then performed to identify CD163 + CD206 + M2 macrophages. (B) M2 macrophages were cultured with the CM from NCI-H226 and NCI-H520 cells infected with LV- HP or LV-Ctrl in the presence or absence of Hb for 24 h. LDH release was analyzed by ELISA. Treated M2 macrophages were treated as in (B) and flow cytometry was used to detect the proportion of (C) 7-AAD positive cells, as well as the mean fluorescence intensity of (D) FerroOrange and (E) C11-BODIPY. (F) Treated M2 macrophages were observed using transmission electron microscopy. Scale bar, 5 µm. Data are presented as the mean ± SD from five independent experiments and were analyzed using an unpaired t-test or a two-way ANOVA followed by a Tukey's post-hoc multiple comparison analysis. ****P<0.0001. ns, not significant; CM, conditioned media; LUSC, lung squamous cell carcinoma; LDH, lactate dehydrogenase; 7-AAD, 7-aminoactinomycin D; HP, haptoglobin.

Article Snippet: An LDH cytotoxicity assay kit (cat. no. HY-K1090; MedChemExpress) was used to assess LDH levels in the supernatant of LUSC cell lines (NCI-H226 and NCI-H520 cells) and M2 macrophages, according to the manufacturer's protocol.

Techniques: Flow Cytometry, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Fluorescence, Transmission Assay, Electron Microscopy, Comparison

Journal: Hereditas

Article Title: RBM15 promotes hypoxia/reoxygenation-induced ferroptosis in human cardiomyocytes by mediating m6A modification of ACSL4

doi: 10.1186/s41065-025-00453-0

Figure Lengend Snippet: Deficiency of RBM15 ameliorated H/R-stimulated oxidative damage in AC16 cells. AC16 cells were treated with H/R, H/R + si-NC or H/R + si-RBM15 and untreated cells were control. ( A and B ) The mRNA and protein levels in AC16 cells were measured by qRT-PCR assay and western blot assay, respectively. ( C ) AC16 cell viability was explored by CCK-8 assay. ( D - G ) The levels of LDH, ROS, MDA and SOD in AC16 cells were examined by related commercial kits. ** P < 0.01, *** P < 0.001

Article Snippet: LDH level in AC16 cells was measured using Promega CytoTox 96 ® kit (Promega, Madison, WI, USA) in line with the protocols.

Techniques: Control, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: Hereditas

Article Title: RBM15 promotes hypoxia/reoxygenation-induced ferroptosis in human cardiomyocytes by mediating m6A modification of ACSL4

doi: 10.1186/s41065-025-00453-0

Figure Lengend Snippet: RBM15 knockdown repressed H/R-induced AC16 cell damage by altering ACSL4 expression. ( A ) The protein level of ACSL4 in AC16 cells transfected with pcDNA-NC or pcDNA-ACSL4 was measured by western blot. ( B - K ) AC16 cells were divided into 5 groups: control, H/R + si-NC, H/R + si-RBM15, H/R + si-RBM15 + pcDNA-NC and H/R + si-RBM15 + pcDNA-ACSL4. ( B ) AC16 cell viability was assessed by CCK-8 assay. ( C - G ) The levels of LDH, ROS, MDA, SOD and Fe 2+ in AC16 cells were examined by commercial kits. ( H ) The protein levels of GPX4, ACSL4, FTH1 and NCOA4 in AC16 cells were measured by western blot. ( I - K ) The levels of GSH, GSSG and GSH/GSSG in AC16 cells were examined by the indicated kits. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: LDH level in AC16 cells was measured using Promega CytoTox 96 ® kit (Promega, Madison, WI, USA) in line with the protocols.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control, CCK-8 Assay