To induce ciliogenesis, cells were serum starved for 24 h. For nutrient deprivation, cells were cultured in HBSS (Thermo Fisher Scientific, #14025-050) for 24 h. To analyse primary cilium response to nutrients, HBSS was supplemented with 0.2, 0.5, 1, 2 and 4 mM l -glutamine (Thermo Fisher Scientific, #25030-024), 20 mM d -(+)-glucose (Sigma-Aldrich, #G7021), 0.5 or 5 mM l -leucine (Sigma-Aldrich, #L8000), or 0.1 or 1 mM l -asparagine (Sigma-Aldrich, #A4159). For AICAR treatment, complete medium was supplemented with 1 mM AICAR (Sigma-Aldrich, #A9978) or dimethyl sulfoxide (DMSO; Sigma-Aldrich, #D2650) for either 24 h (for IF) or 4 h (for western blot analysis). For metformin treatment, complete medium was supplemented with 2 mM metformin (Sigma-Aldrich, #317240) for 4 h (for western blot analysis). For mitochondrial respiratory chain complexes inhibition, cells were treated with 1 µM oligomycin (Agilent Technologies) and 0.5 µM antimycin A/rotenone (Agilent Technologies) for 24 h. For rapamycin treatment, cells were treated with 100 nM rapamycin (LC Laboratories, #R-5000) for 24 h. For asparaginase treatment, cells were treated with 5 U ml−1 asparaginase (Sigma-Aldrich, #A3809) for 24 h.
Asparaginase
Manufactured by Merck Group
Sourced in United States
Asparaginase is a laboratory enzyme that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. It is commonly used in diagnostic and research applications.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Reduced glutathione, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
D5030, by Merck Group (2 mentions)
Recombinant human egf, by Thermo Fisher Scientific (2 mentions)
Dimethyl l glutamate, by Merck Group (2 mentions)
Recombinant il 6, by Merck Group (2 mentions)
D glutamine, by Merck Group (2 mentions)
Stattic, by Santa Cruz Biotechnology (2 mentions)
Azd8055, by Selleck Chemicals (2 mentions)
Dimethyl 2 oxoglutarate, by Merck Group (2 mentions)
Daunorubicin, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Aicar, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Rotenone antimycin a, by Agilent Technologies (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Metformin, by Merck Group (1 mentions)
L asparagine, by Merck Group (1 mentions)
10 protocols using asparaginase
1
Primary Cilium Nutrient Response Modulation
2
Cultured Cell Lines for Cancer Research
3
Quantification of AsnS gene expression
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The total RNAs of the five cells (5×106 cells) without asparaginase (Sigma-Aldrich, St Louis, MO, USA) treatment were extracted according to the manufacturer’s instructions and reverse-transcribed to complementary DNA (cDNA) by using the PrimeScript™ RT Reagent Kit (TaKaRa, Kyoto, Japan). Then, quantitative real-time polymerase chain reaction (qRT-PCR) was performed by using the SYBR® Premix Ex Taq™ II Kit (TaKaRa) on the Roche LightCycler 480 (Roche, Basel, Switzerland). All experiments were conducted in accordance with the manufacturer’s protocols. The primer sequences of AsnS were as follows: forward primer 5′-AAAGTGGAGCCTTTTCTTCCTG-3′ and reverse primer 5′-AGCCAATCCTTCTGTCTGTCATC-3′. Meanwhile, the primer sequences of human GAPDH were as follows: forward primer 5′-CAGCGACACCCACTCCTC-3′ and reverse primer 5′-TGAGGTCCACCACCCTGT-3′. Three parallel analyses were conducted for each sample, and each analysis was repeated three times. The relative expression of the target gene was calculated by inputting the 2−ΔCt values on the LightCycler 480 software (Roche).
4
Proteomic Analysis of Asparaginase Activity
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Standard molecular weight marker proteins were obtained from Bangalore Genei, India. Asparaginase, Diethylaminoethyl (DEAE) cellulose, Sephadex G-100, coomassie brilliant blue R-250, RPMI-1640, fetal bovine serum (FBS), penicillin, streptomycin, 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethylsulphooxide (DMSO), 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI), propidium iodide (PI), rhodamine-123, DNase-free RNase, proteinase-K, phenylmethanesulfonyl fluoride (PMSF), eukaryotic protease inhibitor cocktail, acridine orange and ethidium bromide were procured from Sigma chemical Co., USA. IPG strips were purchased from Bio-Rad Lab., USA. All other chemicals were used of analytical grade and procured from Himedia, Mumbai, India.
5
Metabolic Reprogramming in Cancer Cells
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The following reagents were used: NAC (Sigma-Aldrich, Cat# A7250), tunicamycin (Enzo Life Sciences, Cat# BML-CC104), glycine (VWR, Cat# 10119CU), tBHQ (Sigma-Aldrich, Cat# 19986), ML385 (Sigma-Aldrich, Cat# SML1833), Rapamycin (Sigma-Aldrich, Cat# R0395), Torin 2 (Selleckchem, Cat# S2817), serine (Sigma-Aldrich, Cat# S4500), asparagine (Sigma-Aldrich, Cat# A4159), VEGF-A 165 (R&D, Cat# 293-VE), Asparaginase (Sigma-Aldrich, Cat# A3809), l -Histidinol (Sigma-Aldrich, Cat# H6647), FFA (Sigma-Aldrich, Cat# F9905), α-glycyrrhetinic acid (Sigma-Aldrich, Cat# G10105), A438079 (Sigma-Aldrich, Cat# A9736), Oligomycin A (Sigma-Aldrich, Cat# 75351), CCCP (Sigma-Aldrich, Cat# C2759), antimycin A (Sigma-Aldrich, Cat# A8674), rotenone (Sigma-Aldrich, Cat# R8875), formate (Sigma-Aldrich, Cat# 06473), folate (Enzo Life Sciences, Cat# ALX-460-006). The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500). Primers for qRT-PCR are listed in Supplementary Table 9 .
6
Cultured Cell Lines for Cancer Research
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MDA-MB231 human mammary breast adenocarcinoma (ATCC), HeLa human cervix cancer adenocarcinoma (ATCC) and SiHa human cervix squamous cell carcinoma (ATCC) cells were routinely cultured in DMEM containing 4.5 g/l of glucose and 2 mM of Glutamax, and supplemented with 10% FBS. Assay medium was DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate (D5030, Sigma-Aldrich), reconstituted with 10 mM glucose, 10% FBS, and buffered at pH 7.4 with 3.7 g/L NaHCO3. Cells were grown at 37°C in a humidified 5% CO2 atmosphere. Where indicated, cells were treated with 2 mM of L-glutamine (Invitrogen), 2 mM of dimethyl-L-glutamate (Sigma), 2 mM of dimethyl-2-oxoglutarate (Sigma), 2 mM of D-glutamine (Sigma), 5 mM of reduced glutathione (Merck Millipore),10 nM of rapamycin (Sigma), 15 nM of AZD8055 (Selleckchem), 10-100 ng/ml of recombinant human EGF (PeproTech), 20 ng/ml of recombinant IL-6 (Sigma), 1-10µM of Stattic (Santa Cruz), 10 µM of BPTES or 0.25-1 IU/ml of asparaginase (Sigma). Reagents were dissolved either in DMSO or directly in DMEM according to manufacturer’s indications. When DMSO was used, an equal quantity was also added to control medium. All drugs and reagents were administrated to adherent cells in fresh assay medium.
7
Chemotherapeutic Drugs Cytotoxicity Assay
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The chemotherapeutic drugs, Dex, asparaginase (L-Asp), daunorubicin (DNR), vincristine (VCR), arabinoside (Ara-c), and methotrexate (MTX), were purchased from Sigma. All cells were treated with increasing concentrations of different drugs for 48 h, followed by assessment of cell viability by MTT assay. The IC50 was calculated by linear interpolation.
8
Chemotherapy-Induced T-ALL Cell Apoptosis
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T-ALL cells (10,000 or 40,000 per well) were seeded in 96-well plates and incubated with chemotherapeutic agents at the doses indicated below for 48 h. Chemotherapy doses were as follows unless otherwise indicated: asparaginase, 10 international units/ml (Sigma-Aldrich); dexamethasone, 10 µM (Sigma-Aldrich); vincristine, 1 µM (Selleckchem); doxorubicin, 1 µM (Sigma-Aldrich); etoposide, 10 µM (Sigma-Aldrich); cytarabine, 10 µM (Selleckchem); nelarabine, 10 µM (Sigma-Aldrich); 6-mercaptopurine, 10 µM (Abcam); and methotrexate, 10 µM (Selleckchem). Annexin V and propidium iodide staining were assessed using the Apoptosis Detection kit II (BD Biosciences), and caspase 3/7 activity was assessed using the Caspase Glo 3/7 Assay (Promega) according to the manufacturer’s instructions.
9
Culturing and Characterizing NK Cell Lines
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Natural-killer cell line NK-92 (Cat# CRL-2407) was available from American Type Culture Collection (Manassas, VA, USA). SNK-6 was kindly provided by Professor Norio Shimizu and Yu Zhang of Chiba University. Cell lines were authenticated using Short Tandem Repeat (STR) analysis (Genetic Testing Biotechnology, STR Profile Reports in Supplemental Data). Recent mycoplasma testing has been performed with Lonza LT07-705 MycoAlert™ PLUS Mycoplasma Detection Kit. NK-92 cells were cultured in α-MEM medium supplemented with 10% FBS, 10% HBS and recombinant human IL-2 (20ng/ml). SNK-6 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10% HBS and recombinant human IL-2 (80ng/ml). Asparaginase (Cat# A3809), 13C-glutamine (Cat# 184161-19-1), and glutamine (Cat# 56-85-9) were obtained from Sigma-Aldrich (St.Louis, MO, USA). Anti-PD-1 antibody pembrolizumab (Cat# A2005) was from Selleck Chemicals (Houston, TX, USA).
10
Evaluating Chemotherapeutic Drug Cytotoxicity
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The chemotherapeutic drugs, Dex, daunorubicin, vincristine, arabinoside, methotrexate and asparaginase, were purchased from Sigma. All cells were treated with increasing concentrations of different drugs for 48 h, followed by assessment of cell viability by MTT assay. The IC50 was calculated by linear interpolation.
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