Primary hepatocytes were isolated from mice using the collagenase type X (Wako, Japan; 039-17864) perfusion method.20 (link) Briefly, primary hepatocytes were isolated by collagenase type X (Wako Pure Chemical Industries, ltd, Japan; 039-17864) perfusion method. Cells were washed with hepatocyte wash medium (Thermo Fisher Scientific, Waltham, MA; 17704024), purified by Percoll (GE Healthcare, Chicago, IL; 17089101) density gradient separation, and resuspended in William’s E medium (Thermo Fisher Scientific; 12551032) with 5% fetal bovine serum, 10-nM dexamethasone, and 20-nM insulin. Cells were then seeded on collagen-coated plates at a final density of 3.5 × 104 cells/cm2. After 4 hours, attached cells were cultured with fresh medium and transduced with the indicated adenoviruses. Primary hepatocytes were treated for 4 hours with EBSS (Sigma-Aldrich, St. Louis, MO; E3024), containing 200-μM OA (16 hours; Sigma-Aldrich; O1008), 10-ng/mL TNF (16 hours; PeproTech), 1-μg/mL LPS (6 hours; Sigma-Aldrich; L2630), 50-μM SNAP (4 hours; Sigma-Aldrich, N3398), 100-nM PTIO (30 minutes; Sigma-Aldrich; P5084), and 20-μM CQ (Sigma-Aldrich, C6628). Insulin signaling in primary hepatocytes was stimulated with a 10-minutes exposure to 5-nM insulin (Sigma-Aldrich; I5500).
C6628
Manufactured by Merck Group
Sourced in United States
C6628 is a laboratory equipment product offered by Merck Group. It serves as a general-purpose instrument for various analytical and experimental applications in research and testing environments. The core function of C6628 is to provide a reliable and consistent platform for conducting measurements and analyses, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (4 mentions)
B1793, by Merck Group (4 mentions)
T7402, by Merck Group (2 mentions)
M9281, by Merck Group (2 mentions)
A9434, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bafilomycin, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Hepatocyte wash medium, by Thermo Fisher Scientific (1 mentions)
Collagenase type x, by Fujifilm (1 mentions)
N3398, by Merck Group (1 mentions)
I5500, by Merck Group (1 mentions)
William s e medium, by Thermo Fisher Scientific (1 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)
L2630, by Merck Group (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Importazole, by Cayman Chemical (1 mentions)
Celltiter glo luminescent cell viability assay protocol, by Promega (1 mentions)
38 protocols using c6628
1
Isolation and Treatment of Primary Murine Hepatocytes
2
Pancreatic Cancer Cell Sphere Culture and Xenograft Modeling
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The human pancreatic duct adenocarcinoma cell lines Panc‐1 and MiaPaCa‐2 were obtained from the American Type Culture Collection (Chicago, IL) and cultured in a complete growth medium. The cultured cells were maintained in a 5% CO2 atmosphere at 37°C. The Panc‐1 and MiaPaCa‐2 cell lines were authenticated by checking their gene expressions and were tested for the absence of mycoplasma contamination.
For the pancreatic cancer cell sphere culture, MiaPaCa‐2 cells in RPMI 1640 medium with 0.25% Matrigel were plated into a prepared 96‐well plate with 2% agarose in RPMI 1640 medium (5 cells per well) and cultured for 7‐14 days.
All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Northeast Forestry University. Male immune‐deficient (NPG) mice (4 weeks old) were received from the Beijing Vitalstar Biotechnology Co., Ltd. (Beijing, China) and fed in a sterile environment with food and water ad libitum. For xenografts, 2 million cells suspended in 100 μL of PBS were subcutaneously injected into the lower flank of the NPG mice. The cell‐transplanted mice received TM (500 μmol/L; 323446 Sigma, St. Louis, MO, USA) or chloroquine diphosphate salt (CQ; 100 μmol/L; C6628 Sigma) individual treatments, or TM (500 μmol/L) combined with CQ (100 μmol/L).
For the pancreatic cancer cell sphere culture, MiaPaCa‐2 cells in RPMI 1640 medium with 0.25% Matrigel were plated into a prepared 96‐well plate with 2% agarose in RPMI 1640 medium (5 cells per well) and cultured for 7‐14 days.
All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Northeast Forestry University. Male immune‐deficient (NPG) mice (4 weeks old) were received from the Beijing Vitalstar Biotechnology Co., Ltd. (Beijing, China) and fed in a sterile environment with food and water ad libitum. For xenografts, 2 million cells suspended in 100 μL of PBS were subcutaneously injected into the lower flank of the NPG mice. The cell‐transplanted mice received TM (500 μmol/L; 323446 Sigma, St. Louis, MO, USA) or chloroquine diphosphate salt (CQ; 100 μmol/L; C6628 Sigma) individual treatments, or TM (500 μmol/L) combined with CQ (100 μmol/L).
3
Chloroquine Inhibits Autophagic Flux
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Chloroquine (C), an anti-inflammatory substance, is the most commonly used autophagy completion inhibitory drug to ascertain autophagic flux because of its suitability in vivo [31 (link)]. HT29 cells were treated with chloroquine (10 μM; C6628 Sigma-Aldrich, Budapest, Hungary; diluted in DMSO) for 1 h before treatments with DNA. All treatments were performed in triplicate. Between plates, two samples received the same treatment to avoid possible manual errors in the treatments between plates.
The treatment plan for HT29 cells is shown inTable 1 .
The treatment plan for HT29 cells is shown in
4
Comprehensive Autophagy and Nuclear Transport Inhibitor Protocol
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Purchased compounds included chemotherapeutics [paclitaxel (Sigma #T7402)], early stage autophagy inhibitor [PI-103 (LC Labs #P-9099)], late-stage autophagy inhibitors [bafilomycin A1 (Sigma #SML1661, LC Labs #B-1080 in LC3B puncta and autophagic flux assays, and Cayman Chemical #11038 for cytotoxicity assays)] and chloroquine (Sigma #C6628), and nucleocytoplasmic transport pathway inhibitors [importazole (Cayman Chemical #21491) and KPT-330 (Cayman Chemical #18127)] [52 (link), 58 (link)]. Structures of non-FDA-approved small molecules may be found here: bafilomycin A1 [59 ], importazole [60 (link)], and HTP-013 [12 (link)]. Photoaffinity labeled PHY analogs were created as previously published [17 (link), 61 (link)]. All compounds were suspended in dimethyl sulfoxide (DMSO), and the final vehicle concentration < 0.1% (v/v).
5
Cell Viability Assay with CQ and ULK1 Inhibitor
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Cells were plated in sealed white 96-well plates (2000 cells/well). The next day, the cells were treated with compounds, CQ (Sigma-Aldrich, C6628) or ULK1 inhibitor (Sigma-Aldrich, SML1540) as indicated, and luminescence was measured at the indicated time points using the CellTiter-Glo® Luminescent Cell Viability Assay protocol (Promega, G7573).
6
Neurotoxicity of HIV-infected Macrophages
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Neuronal cultures were maintained until DIV 21. At DIV21-22, neurons were treated with Mock/MDMs or HIV/MDMs. Cells were treated with the same supernatants from the same donor (ND527) unless otherwise stated. For experiments with different sets from different donors, we titrated every supernatant set to 50% neurotoxicity to ensure that we were eliciting the same toxic insult. To mimic excitotoxicity, cells were treated with NMDA (Tocris, Bioscience, Bristol, United Kingdom, 0114) or vehicle (H2O). The dilutions used for each set of HIV/MDM supernatants was determined by titrating to 50% neurotoxicity as quantified by immunohistochemistry for MAP2. Chloroquine (CQN; 1 or 2 μM; C6628, Sigma Aldrich, St. Louis, MO, USA) or vehicle (DMSO; 276855, Millipore Sigma, Burlington, MA, USA) treatment was performed 4 h prior to treatment with Mock/MDMs, HIV/MDMs, or NMDA (5, 10, 15 and 20 μM). Treatment with MG132 (1 or 5 μM; 10012628, Cayman Chemical, Ann Arbor, MI, USA), MK-801 (1 μM; 0924, Tocris Bioscience, Bristol, United Kingdom) and their respective vehicles (DMSO and H2O) was performed 1 h prior to treatment.
7
Cockroach Allergen-Induced Asthma Model
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The cockroach allergen–induced asthma mouse model was established with the protocol as illustrated in Figure 1A
8
Autophagy Modulation of Bluetongue Virus
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Three drugs, 3-methyladenine (3-MA) (M9281, Sigma), chloroquine (CQ) (C6628, Sigma) and rapamycin (Rapa) (S1842, Beyotime), were used in this experiment to explore the effects of autophagy on the replication of BTV. The cytotoxicity of these drugs at optimal concentrations (10 mM 3-MA; 20 μM CQ; 100 nM Rapa) was tested using a WST-1 cell proliferation and cytotoxicity assay kit (C0035, Beyotime) according to the manufacturer’s protocol. Next, BSR cells were pretreated with optimal concentrations of Rapa, 3-MA for 2-4 h and were not pretreated with CQ prior to BTV infection. Subsequently, the cells were infected with BTV1 as above and further incubated in fresh media in the absence or presence of these drugs at the same concentrations as for the pretreatments. Corresponding DMSO or ddH2O were used as vehicle controls. At the indicated times, the virus from the supernatants and cells were collected and the viral protein expression were detected as above.
9
Evaluating SOX2 Knockdown and Chemoresponse in Xenograft Mice
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SW620 cells stably transfected with SOX2 short hairpin RNA (shRNA) or negative shRNA (5 × 106 cells in 0.1 mL PBS) were subcutaneously injected into the right dorsal flanks of 4-week-old female Balb/c nude mice (n = 3/group), respectively. Twenty-two days later, mice bearing tumors were administered intraperitoneal injection of PBS, Chloroquine (Sigma-Aldrich, C6628, CQ, 20 mg/kg), irinotecan (Sigma-Aldrich, I1406, 20 mg/kg), or combination of irinotecan and CQ every 3 days. Tumor size was measured with a digital caliper every 3 days. Tumor volume was calculated from digital caliper measurements of tumor dimensions in mm using the formula for a prolate ellipsoid: (L × W2)/2, where L is the longer of the 2 measurements. At the endpoint, tumors were harvested and weighed. The excised tissues were either fixed in 10% neutral-buffered formalin or snap frozen in liquid nitrogen. Tumor sections from paraffin-embedded blocks were used for histologic examination. All animal experiments were conducted as per the protocol approved by the Animal Care and Use Committee of the Southern Medical University.
10
Cellular Density Determination under Stress
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Cells were seeded in a 24-well culture plate (Greiner Bio One, 662160) and incubated under normal, serum starved or hypoxic conditions (0.2% O2). CQ was used at a concentration of 5 µg/ml (Sigma Aldrich, C6628). Cellular density was determined using Cristal violet staining.
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