ros detection cell-based assay kit (dcfda)  (Cayman Chemical)

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis

doi: 10.3390/ijms25021157

Figure Lengend Snippet: Orf7b expression activated cleaved caspase 8 but decreased GPX4. ( A – C ) Immunoblotting revealed upregulation of cleaved caspase 8 ( A ), no change in expression level for caspase 9 ( B ) and downregulation of GPX4 ( C ), with corresponding densitometric plots. ( D ) The MDA assay revealed that Orf7b induced statistically significant lipid peroxidationin Beas-2B cells compared with the control group. ( E ) The DCFDA assay revealed that there was significant increase in intracellular ROS levels in the Orf7b group compared with the control group. ( F , G ) Concentration response-based apoptosis inhibitor (Z-VAD-FMK) and ferroptosis inhibitor (Ferrostatin) studies using LDH assays showed partial alleviation of cell death in Orf7b-overexpressed Beas-2B cells, respectively. ( H ) A ferroptosis inhibitor, Liproxstatin-1, based inhibitor study confirmed the involvement of the ferroptosis pathway. For ( A – C )—C: control group, Orf7b: Orf7b overexpressed group.*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ns: not significant. p values between two groups were calculated using a student t -test. p values between three or more groups were calculated using a one-way ANOVA. The data are from three independent biological experiments.

Article Snippet: ROS Detection Cell-Based Assay Kit (DCFDA) was purchased from Cayman Chemical, Ann Arbor, MI, USA (Cat# 601520).

Techniques: Expressing, Western Blot, Multiple Displacement Amplification, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis

doi: 10.3390/ijms25021157

Figure Lengend Snippet: Orf7B upregulates c-Myc to modulate the activation of cleaved caspase 8 and the reduction of GPX4. ( A – D ) Immunoblotting revealed upregulation of c-Myc in Orf7b-transduced Beas-2B cells ( A ), upregulation of TNF-α in Orf7b-transduced Beas-2B cells ( B ). CRISPR/CAS9 knockout of c-Myc in Orf7b-transduced Beas-2B cells ( C ) implicated c-Myc as the critical regulator in apoptosis and ferroptosis by downregulating the expression of cleaved caspase 8 ( D ) and alleviating the expression of GPX4 ( E ). Corresponding densitometric plots for the immunoblots confirm the results ( A – E ). ( F ) The LDH assay confirmed the attenuation of cell death post c-Myc knockout. ( G ) The DCFDA assay revealed a decrease in intracellular ROS levels after knocking out c-Myc. For ( A , B , D )—C: Control, 7b: Orf7b overexpression, M: c-Myc knockout + Orf7b overexpression. For the densitometric plots—C: control group, Orf7b: Orf7b overexpressed group *: p ≤ 0.05, **: p ≤ 0.01. p -values between the two groups were calculated using a student t -test. p -values between three or more groups were calculated using a one-way ANOVA. The results are representative of three independent experiments.

Article Snippet: ROS Detection Cell-Based Assay Kit (DCFDA) was purchased from Cayman Chemical, Ann Arbor, MI, USA (Cat# 601520).

Techniques: Activation Assay, Western Blot, CRISPR, Knock-Out, Expressing, Lactate Dehydrogenase Assay, Over Expression

Journal: European journal of cell biology

Article Title: HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

doi: 10.1016/j.ejcb.2022.151271

Figure Lengend Snippet: Effects of TS and gp120 exposure on cell viability and oxidative stress. A : MTT cytotoxicity assay for cell viability following TS - gp120 treatments for 24 h. B : DCFDA assay as an indicator for reactive oxygen species (ROS) in cells. n = 3 biological replicates/group. *p < 0.05, ** p < 0.01, **p < 0.001, ****p < 0.0001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TS + gp120 co-treatment. n.s. = non statistical significance.

Article Snippet: HBMECs were seeded on 96 well plates and treated for 24 h. Intracellular ROS was assessed by employing ROS Detection Cell-Based Assay Kit – DCFDA (Cayman Chemical # 601520) as per the protocol mentioned therein.

Techniques: Cytotoxicity Assay