ldh cytotoxicity assay kit  (TaKaRa)

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Baicalein Protects H9c2 Cardiomyoblasts Against LPS-Induced Inflammatory Injury by Modulating the NF-κB/NLRP3 Inflammasome Pathway and Mitochondrial ROS

doi: 10.5812/ijpr-169689

Figure Lengend Snippet: Baicalein preserves cell viability and morphology in lipopolysaccharide (LPS)-challenged H9c2 cardiomyoblasts. A, MTT cell viability assay. H9c2 cells were seeded at 1 × 10⁴ cells/well in 96-well plates and pretreated with baicalein (5 - 20 µM; 1 h), followed by LPS (1 µg/mL; 24 h). MTT reagent (5 mg/mL, Sigma M2128) was added for 4 h, formazan crystals were dissolved in DMSO, and absorbance was recorded at 570 nm (BioTek Synergy HTX). A calibration curve (1 – 8 × 10³ cells/well) was generated to validate linearity. Bars show mean ± SD. B, Inflammasome transcript kinetics (0 - 4 h). Total RNA was extracted using TRIzol™ (Invitrogen), converted to cDNA using the High-Capacity kit, and qPCR was performed with SYBR™ Green on a QuantStudio™ 5. Nlrp3, Il1b, and Il18 expression was evaluated from 0 - 4 h after LPS exposure to identify the mechanistic transcriptional peak. Relative abundance was calculated via 2⁻ΔΔCt using GAPDH as the reference. C, LDH release assay for membrane integrity. Supernatants were collected after 24 h of LPS stimulation, and LDH activity was quantified using the Takara MK401 kit. Absorbance at 490 nm was compared with the maximum lysis control to compute LDH release (%). D, Phase-contrast morphology. H9c2 cells were imaged on an Olympus CKX53 inverted microscope (20× objective) to assess rounding, shrinkage, and detachment following LPS exposure, and morphological preservation following baicalein pretreatment.

Article Snippet: - Lipopolysaccharide (LPS, E. coli O111:B4): Sigma-Aldrich, Cat. No. L2630 - Baicalein (≥ 98% purity): Sigma-Aldrich, Cat. No. 465119 - N-acetyl-L-cysteine (NAC): Sigma-Aldrich, Cat. No. A9165 - MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide): Sigma-Aldrich, Cat. No. M2128 - LDH Cytotoxicity Detection Kit: Takara Bio, Cat. No. MK401 - JC-1 Mitochondrial Membrane Potential Assay Kit: Abcam, Cat. No. ab113850 - DCFH-DA ROS Detection Probe: Sigma-Aldrich, Cat. No. D6883 - TRIzolTM Reagent: Invitrogen, Cat. No. 15596026 - High-Capacity cDNA Reverse Transcription Kit: Applied Biosystems, Cat. No. 4368814 - SYBRTM Green PCR Master Mix: Applied Biosystems, Cat. No. 4367659 - Enzyme-linked immunosorbent assay (ELISA) Kits: Rat IL-1β ELISA: Abcam, Cat. No. ab255730

Techniques: Viability Assay, Generated, SYBR Green Assay, Expressing, Lactate Dehydrogenase Assay, Membrane, Activity Assay, Lysis, Control, Inverted Microscopy, Preserving

Journal: Biomolecules

Article Title: Albumin Protects Against Cyclophosphamide-Induced Hemorrhagic Cystitis by Scavenging Acrolein and Reactive Oxygen Species

doi: 10.3390/biom16040536

Figure Lengend Snippet: Alb prevents urothelial cell injury in vitro. ( A – C ) Alb protects against ACR (ACR)-induced cytotoxicity. Cultured urothelial cells were treated with ACR (100 µM) in the presence or absence of Alb at the indicated concentrations. Cell injury/viability was assessed by AM/PI live–dead staining ( A ), formazan formation assay ( B ), and lactate dehydrogenase (LDH) release ( C ). Data are mean ± SE ( n = 3). ** p < 0.01 vs. untreated control; ## p < 0.01 vs. ACR alone. ( D – F ) Alb preserves ferroptosis-defense proteins following ACR exposure. Cells were treated with ACR (50 µM) with or without Alb (30 mg/mL). After 9 h, cell lysates were analyzed by Western blotting for GPX4 and xCT. ( D ) Representative immunoblots; ( E , F ) Densitometric quantification. Data are mean ± SE ( n = 3). * p < 0.05, ** p < 0.01 vs. −ACR control; # p < 0.05 and ## p < 0.01 vs. ACR alone (as indicated). ( G , H ) Alb protects against H 2 O 2 -induced injury. Cells were treated with the indicated concentrations of H 2 O 2 in the presence or absence of Alb (30 mg/mL). Cell injury/viability was assessed using the same assays as in ( A – C ).

Article Snippet: LDH release was measured using the LDH Cytotoxicity Detection Kit (TaKaRa Biomedicals, Otsu, Japan) following the manufacturer’s protocol.

Techniques: In Vitro, Cell Culture, Staining, Tube Formation Assay, Control, Western Blot