MDCK cells (100,000 cells/well, 2 mL/well) were seeded into 6-well plates, and the plates were incubated overnight at 37 °C and 5% CO2. Lung tissues were homogenized in 1 mL of VP medium, and the homogenate was centrifuged for 10 min at 12,000 rpm. The supernatants were then diluted with VP medium supplemented with TPCK-treated trypsin (2 µg/mL; Merck, Germany) and 1% PS solution (Thermo Fisher Scientific, USA) to an initial dilution of 1:10, which was later serially diluted by tenfold. When the MDCK cells reached above 90% confluence, PBS was added to rinse the plates twice, after which infection was carried out using each dilution of the supernatant (200 µL). Following the 1-hour incubation at 37 °C (under shaking every 20 min), samples were eliminated and substituted by VP medium (2 mL) containing TPCK-treated trypsin (2 µg/mL; Merck, Germany), 1% PS solution (Thermo Fisher Scientific, USA), and 1.6% agarose (Lonza, Swiss). The plates were then incubated for 72 h at 37 °C and 5% CO2. Next, the neutral red staining solution (Beyotime, China) was added to all wells, and the plates were incubated for another 4 h at 37 °C and 5% CO2. Finally, the staining solution was discarded, and the viral plaques were counted. The lung viral load was calculated and expressed as plaque forming unit (PFU) per millilitre.
Neutral red staining solution
Manufactured by Beyotime
Sourced in China
Neutral red staining solution is a laboratory reagent used for the visualization and differentiation of various cellular structures and components in biological samples. It is a commonly used dye that selectively stains living cells and organelles, allowing for the identification and analysis of cell viability, morphology, and other cellular characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Agarose, by Lonza (1 mentions)
Tpck treated trypsin, by Merck Group (1 mentions)
Dextran standard, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Galactose, by Merck Group (1 mentions)
Fucose, by Merck Group (1 mentions)
Rhamnose, by Merck Group (1 mentions)
Mannose, by Merck Group (1 mentions)
Xylose, by Merck Group (1 mentions)
Arabinose, by Merck Group (1 mentions)
Pd98059, by Selleck Chemicals (1 mentions)
Nitric oxide assay kit, by Beyotime (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Anti p65, by ABclonal (1 mentions)
Anti erk, by ABclonal (1 mentions)
Anti p38 mapk, by ABclonal (1 mentions)
Anti phospho erk, by ABclonal (1 mentions)
Anti phospho jnk, by ABclonal (1 mentions)
Anti phospho akt, by ABclonal (1 mentions)
Anti jnk, by ABclonal (1 mentions)
6 protocols using neutral red staining solution
1
MDCK Viral Plaque Assay for Lung Samples
2
Hericium erinaceus Extract Bioactivity Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fruiting body of Hericium erinaceus was purchased from Jilin Jiaohe Songshan Food Co., Ltd. (Jiaohe, China). Dextran standards (670, 410, 270, 80, 25, 12 and 5 kDa) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monosaccharide standards, including arabinose, mannose, fucose, xylose, rhamnose, galactose and glucose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cell Counting Kit-8 was obtained from APExBIO Technology LLC (Houston, TX, USA). Neutral red staining solution, nitric oxide assay kit and BCA kit were obtained from Beyotime Biological Technology Co., Ltd. (Shanghai, China). Western Blot assay kit, SDS-PAGE kit, ELISA kits of TNF-α, IL-6, IL-10 and IFN were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China). Anti-ERK, anti-phospho ERK, anti-JNK, anti-phospho JNK, anti-p38 MAPK, anti-phospho p38 MAPK, anti-Akt, anti-phospho Akt, anti-IkBα, anti-p65, anti-β-actin and anti-Histone H2A were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China). Inhibitors of BAY11-7082, SB203580, SP600125, PD98059 and LY294002 were purchased from Selleck Chemicals (Shanghai, China).
3
Inflammatory Signaling Pathway Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SCSP was provided by Wuxi MimoTopes Biotechnology (Wuxi, China) [20 (link)]. CTX was purchased from Hengrui Medicine (Lianyungang, China). The DAB immunohistochemistry kit was purchased from Boster (Wuhan, China). Neutral red staining solution and primary antibodies against β-actin, NF-κB p50, NF-κB p65, IKKα, IKKβ, and IκBα were supplied by Beyotime (Shanghai, China). The remaining primary antibodies were provided by Cell Signaling Technology Inc. (Beverly, MA, USA).
4
H1N1 Virus Titration in MDCK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1N1 influenza virus was infected into MDCK-traf3−/− and wild-type MDCK cells at MOI 0.01. The culture supernatants were harvested at 24 h, 48 h, 72 h and 96 h after infection and used for plaque assay to determine the virus titers. The supernatant was diluted 10 times gradually before being added to a 6-well culture plate containing MDCK cells. One hour later, the supernatant was discarded and then the solution containing 1 μg/mL TPCK-trypsin (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin liquid (Gibco, Grand Island, NY, USA) was prepared by mixing 2 × DMEM medium into an equivalent microcrystalline cellulose solution. After culturing for an additional 96 h, the live cells were stained with 1 mL neutral red staining solution (Beyotime, China) to observe plaque formation.
5
Osteogenic Differentiation Assay with VK2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in 6-well plates in complete medium. After the cells reached 80~90% confluence, the medium was replaced by the osteogenic medium with different concentrations of VK2 for 7 days. Then, the cells were fixed for 20 min with 4% paraformaldehyde, and ALP staining was performed using the BCIP/NBT alkaline phosphatase chromogenic kit following the manufacturer’s instructions (Beyotime Biotechnology, Beijing, China). Cells were observed under a light microscope (Nikon, Tokyo, Japan) after staining by neutral red staining solution (Beyotime Biotechnology) for 5 min.
After treatment with VK2 for 7 days, the cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime Biotechnology). Cell lysates were analyzed for protein concentration using the BCA protein concentration assay kit (Beyotime Biotechnology). Then, ALP activity was measured using an ALP assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
After treatment with VK2 for 7 days, the cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime Biotechnology). Cell lysates were analyzed for protein concentration using the BCA protein concentration assay kit (Beyotime Biotechnology). Then, ALP activity was measured using an ALP assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
6
Mechanism of BDE-47-induced Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BDE-47 (≥99.9%) was obtained from CSNpharm (Chicago, IL, USA). ISL (≥98%, HPLC) and LCB (≥98%, HPLC) were obtained from Yuanye (Shanghai, China). DCFH-DA and DMSO were obtained from Solarbio (Beijing, China). An apoptosis kit was obtained from Multi science (Hangzhou, China). A DAPI dye and neutral red staining solution were obtained from Beyotime Biotechnology (Shanghai, China). ELISA kits were obtained from Mlbio (Shanghai, China) (TNF-α Cat. No. ml002095; IL-6 Cat. No. ml063159; IL-1β Cat. No. ml301814). CAT, SOD, and GSH kits were obtained from Solarbio (Beijing, China). β-Actin, Nrf2, Keap1, HO-1, NQO1, IKBKB, IκB-Alpha, Bax, Bcl-2 antibodies, and the ECL luminescent reagent were obtained from Proteintech (Wuhan, China) (β-Actin Cat. No. 66009-1-lg; Nrf2 Cat. No. 16396-1-AP; Keap1 Cat. No. 60027-1-lg; HO-1 Cat. No. 66743-1-lg; NQO1 Cat. No. 67240-1-lg; IKBKB Cat. No. 15649-1-AP; IκB-Alpha Cat. No. 10268-1-AP; Bax Cat. No. 50599-2-lg; Bcl-2 Cat. No. 26593-1-AP). Caspase-3 antibody was obtained from BBI (Shanghai, China) (Cat. No. D16009). NF-κB and p-NF-κB antibodies were obtained from Abcam (Cambridge, UK) (NF-κB Cat. No. ab76302; p-NF-κB Cat. No. ab32536). P-Nrf2 antibody was obtained from Abclonal (Wuhan, China) (Cat. No. AP1133). Real-time fluorescent quantitative reagents were obtained from TransGen Biotech (Beijing, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!