F98 and C6 rat glioblastoma, human glioma U87 MG, and human embryonic kidney 293T cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA), while the human glioma cell lines U251 and SHG44 were purchased from Institute of Basic Medical of Science, Research Chinese Academy of Medical Sciences. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HyClone, UT, USA) in a humidified atmosphere with 5% CO2 and 95% air at 37°C. Puromycin (Invitrogen, USA), rapamycin (Invitrogen), Chloroquine (CQ; Invitrogen) and 3-methyladenine (3-MA; Sigma–Aldrich, USA) were dissolved in DMSO prior to use.
Chloroquine cq
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chloroquine (CQ) is a laboratory reagent used in various scientific applications. It is a chemical compound with the molecular formula C₁₈H₂₆ClN₃. Chloroquine serves as a core function in research and analysis, without further interpretation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
3 methyladenine 3 ma, by Merck Group (3 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
U87mg, by American Type Culture Collection (1 mentions)
Streptomycin, by Welgene (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Ecl solution, by Merck Group (1 mentions)
3 4 5 dimetylthiazol 2 yl 2 5 diphenyltetrazoliumbromide mtt, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Welgene (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Welgene (1 mentions)
Ly294002, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Penicillin, by Welgene (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Bafilomycin a1 bfa, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Mcf 7, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Agm bulletkit, by Lonza (1 mentions)
9 protocols using chloroquine cq
1
Cell Culture Protocol for Glioblastoma and Embryonic Kidney
2
In Vitro Cellular Assays for Drug Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GJ powder provided by Hanpoong Pharmaceutical Co. (Jeonju, Korea) was dissolved in D.W. LY294002, SC79, 3-methyladenine (3-MA), 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and chloroquine (CQ) was from Invitrogen (San Diego, CA, USA). ECL solution were obtained from Merck Millipore (Middlesex, MA, USA) and Z-VAD-FMK was provided by R&D Systems, Inc. (Northeast, MN, USA), Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI1640), penicillin and streptomycin were obtained from WELGENE (Gyeongsan, Korea). Fetal bovine serum (FBS) was obtained from GR scientific (Bedford, UK).
3
Monocyte Autophagy Regulation in Leprosy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells from healthy donors were isolated via Ficoll-Paque PLUS method (GE Healthcare, 17-1440-03) under endotoxin-free conditions. Cells were ressuspended in RPMI 1640 supplemented with 10% FBS, 2 mM l -alanyl-l -glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, P4333) and plated at 5 × 105 cells/mL on 15-mm sterile circular coverslips and cultured for 2 h at 37°C in a 5% CO2 atmosphere. Alternatively, cells were plated at 1 × 106 cells/mL in 24-well plates for qPCR assays. The supernatant was discarded and coverslips were rinsed with PBS to remove non-adherent cells. The media was replaced and the monocytes were stimulated with armadillo γ-irradiated M. leprae at 10 µg/mL (~10:1) in the presence or absence of the following stimuli for 18 h at 37°C in a 5% CO2 atmosphere. Autophagy was triggered with 200 ng/mL rapamycin (RP) (Sigma-Aldrich, R0395), 100 µM chloroquine (CQ) (Invitrogen, P36235) was used as an autophagic flux blocker, and 10 µM 3-methyladenine (3-MA) (Sigma-Aldrich, M9281) as an autophagy inhibitor.
4
Vomocytosis Inhibition Drug Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For drug-treated experimental groups, DCs were infected with CN at a 5:1 c.p.r. using identical methods outlined previously. After the 2-hour phagocytosis and washing step, the wells were treated with DC media containing either 10 uM of chloroquine [CQ] (Thermo Fisher Scientific), 100 nM of cytochalasin B from Drechslera dematioidea [CYT low] (Sigma), 4 uM of cytochalasin B (CYT hi), or 100 nM of bafilomycin A1 [BFA] (Sigma). These parameters were selected based on the range of drug concentrations used in prior vomocytosis studies[16 (link), 18 (link), 24 (link), 46 (link)]. This step was followed by time-lapse imaging for 14 hours whilst in drug-containing media.
5
Chemo-Sensitization of Glioblastoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Compound C (6‐[4‐[2‐(1‐Piperidinyl)ethoxy]phenyl]‐3‐(4‐pyridinyl)pyrazolo[1,5‐a]pyrimidine dihydrochloride: Dorsomorphin dihydrochlo ride) was purchased from Abcam. TMZ, carmustine (1,3‐bis(2‐chlorethyl)‐1‐nitrosourea; BCNU) and U0126 were purchased from Sigma. Chloroquine (CQ) was purchased from Thermo Fisher Scientific. Human GB cell lines, U251 (U251MG‐Luc, JCRB1386) and A172 (A‐172, JCRB0228), and the human metastatic mammary carcinoma cell line MCF7 (MCF7, JCRB0134) was purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The U251 cell line was engineered to express the firefly luciferase gene. The human GB cell line T98 (T98‐G, CRL‐1690) was purchased from the ATCC. The human pancreatic cancer cell line PANC1 (PANC‐1, RCB2095) was purchased from RIKEN BioResource Research Center (RIKEN BRC). Normal human astrocytes (NHA) were purchased from the Lonza group. In all cases, early‐passage cultures were stored and used for the experiments. The GB cell lines were cultured in DMEM (Sigma‐Aldrich) containing 10% FBS and 1% penicillin‐streptomycin. MCF7 and PANC‐1 were cultured in RPMI‐1640 with L‐glutamine and phenol red medium containing 10% FBS and 1% penicillin‐streptomycin. The normal human astrocytes (NHA) were cultured in specialized medium (AGM BulletKit) purchased from the Lonza group. D‐Luciferin was purchased from Promega (Madison, WI, USA).
6
Quantitative Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated in a 24-well plate at a density of 7.5 × 105 cells/well, and allowed to adhere for at least 24 h. Subsequently, the cells were starved for 3 h with DMEM (0.5% FBS) before treatment, and then treated with 30 µM chloroquine (CQ) (Molecular Probes, Invitrogen, Eugene, OR, USA) for 16 h. Next, the cells were incubated with formaldehyde 3.7% in Dulbecco’s phosphate-buffered saline (DPBS 1X) (Sigma-Aldrich) and permeabilized with 0.2% Triton X-100 in DBPS 1X for 15 min at room temperature. The cells were incubated for 2 h with a primary LC3 rabbit polyclonal antibody (Molecular Probes, Invitrogen, Eugene, OR, USA) diluted in DPBS with 5% BSA (Bovine Serum Albumin), followed by the secondary antibody Alexa Fluor 488 (Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Finally, the cells were labeled with HOECHST 33342 (1:2000) (Life Technologies) and phalloidin-rhodamine (1:200) (Molecular Probes, Invitrogen, Eugene, OR, USA). Images were acquired by the High Content In Cell Analyzer 2200 platform (GE Healthcare Life Sciences, Chicago, IL, USA) and quantification was performed in the Image-Pro software (Media Cybernetics, Rockville, MD, USA).
7
Imaging and Quantifying Mitophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to visualize mitochondrial engulfment in autophagic vacuoles we performed a co-labeling with LysoTracker Green (LTG) and MTRC. For confocal live imaging, cells were grown on MatTek glass bottom chambers and stained with LTG 200nM for 45 minutes at 37°C. During the last 15 min of incubation, cells were exposed to MTRC 200nM and then analyzed after 30 min using a Leica TCS SP5 II confocal microscope. Colocalization analyses were performed using JACoP plugin in ImageJ [36 (link)]. Pearson's correlation coefficient (PCC) was used as the parameter to measure co-localization in our samples.
The mitophagic flux was quantified by flow cytometry. Briefly, HT22 cells were stained with 50nM Mitotracker Deep Red (MTDR; Molecular Probes) for 15 min at 37°C and treated for 1h with 30 μM of the lysosomal inhibitor chloroquine (CQ, Molecular Probes) [37 (link)]. The MTRD MFI was evaluated before and after the treatment with CQ, and mitophagic flux was quantified as the ratio of the MTDR fluorescence w/o CQ.
The mitophagic flux was quantified by flow cytometry. Briefly, HT22 cells were stained with 50nM Mitotracker Deep Red (MTDR; Molecular Probes) for 15 min at 37°C and treated for 1h with 30 μM of the lysosomal inhibitor chloroquine (CQ, Molecular Probes) [37 (link)]. The MTRD MFI was evaluated before and after the treatment with CQ, and mitophagic flux was quantified as the ratio of the MTDR fluorescence w/o CQ.
8
Combination Treatment Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
6-shogaol (≥90%), Taxol (≥95%), and DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) (≥98%) were purchased from Sigma. Chloroquine (CQ) was from Molecular Probes, Invitrogen. Fluoromount G was procured from Electron Microscopy Sciences. DAPI (4',6-Diamidino-2-Phenylindole), Giemsa and other fine chemicals were from Sigma. Chemiluminescent western blotting detection system was from Thermo Scientific. FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen (Cat # 556547). Ultra low attachment plates were obtained from Corning, USA and MEBM (Mammary Epithelial Basal Media) was procured from Lonza, USA.
9
Immunofluorescence Imaging of Mitophagy
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!