All cells were purchased from the American Type Culture Collection (ATCC, Barcelona, Spain). HT-29 (colorectal adenocarcinoma), HTB-54 (grade III lung carcinoma), MCF7 (mammary adenocarcinoma), PC-3 (grade IV prostate adenocarcinoma) and K-562 (chronic myelogenous leukemia) were cultured in RPMI (Gibco, Madrid, Spain), 10% FBS (Gibco), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco). BEAS-2B cell line (normal epithelial lung) was cultured in DMEM (Gibco), 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. 184B5 (normal mammary gland) cell line was cultured in DMEM:F12 supplemented with 5% FBS, 1 × ITS (Lonza, Barcelona, Spain), 100 nM hydrocortisone (Sigma-Aldrich), 2 mM sodium pyruvate (Lonza), 20 ng/mL EGF (Sigma-Aldrich), 0.3 nM trans-retinoic acid (Sigma-Aldrich), 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were cultured at 37 °C under 5% CO2.
Trans retinoic acid
Manufactured by Merck Group
Sourced in United States, Spain, United Kingdom
Trans-retinoic acid is a chemical compound that is used in various laboratory applications. It is a type of retinoid, a class of organic compounds derived from vitamin A. Trans-retinoic acid serves as a research tool for studying biological processes and cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Sodium pyruvate, by Lonza (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Htb 54, by Thermo Fisher Scientific (2 mentions)
Ht 29, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
L alanyl l glutamine, by Thermo Fisher Scientific (2 mentions)
Sh sy5y line, by European Collection of Authenticated Cell Cultures (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Metamorph microscopy automation and image analysis software, by Molecular Devices (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (2 mentions)
34 protocols using trans retinoic acid
1
Cell Culture Protocol for Cancer Research
2
Neuronal Differentiation and Purification
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We followed a procedure adapted from Pleasure (Pleasure et al. 1992 (link)). Neural differentiation was induced in NTERA-2 cells by supplementing the growth medium with 0.01 mM trans-retinoic acid (Sigma-Aldrich, St. Louis, MO, USA) for at least 4 weeks. Neuronal and glial NTERA-2 cells were selectively detached and replated by using 0.02% trypsin (Gibco) and delivering 10 hard taps to both sides of the T75 culture flask. Replated cells were maintained with growth medium supplemented with the following mitotic inhibitors: 1 μM cytosine arabinoside (Sigma-Aldrich), 10 μM fluoro-deoxyuridine (Sigma-Aldrich) and 10 μM uridine (Sigma-Aldrich). Differentiated cells were maintained with mitotic inhibitors for at least 2 weeks before the introduction of labeled monosaccharides.
3
Differentiation and Infection of HBEC Cultures
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Human bronchial epithelial cells (HBEC) were purchased from Lonza and expanded in Bronchial Epithelial cell Growth Medium (BEGM) supplemented with the recommended additives (Singlequots, Lonza) until 80% confluent (P0 cells). Once confluent the cells were aliquoted and frozen down as ‘P1’ cells. For each experiment P1 cells were expanded to 80% confluence and seeded onto a 12-well Transwell permeable support (0.4 µM, Corning, NY) in BEGM media diluted 1∶1 in DMEM (Lonza, Invitrogen) and supplemented with BEGM Singlequots devoid of triiodothyronine (T3) and retinoic acid (differentiation media). All trans-retinoic acid (Sigma, 50 nM in ethanol) was added back fresh for each media exchange. Cells were infected by either RSV-A2-GFP [31] (link), [32] (link), or a clinical isolate designated RSV-A2-MOT0972 (Obtained from Edward Walsh, University of Rochester Medical School) at various multiplicity of infection (MOI), as indicated in the text. Six days post-seeding the media was removed from the apical side and cells were cultured at ALI for an additional 14 days. Media was replaced three times a week with 1 mL on the basal side and for week one, 400 µL was added on the apical side.
4
Neuroblastoma cell line UPR activation
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SK-N-SH, a human neuroblastoma cell line (European Collection of Cell Cultures #86012802, Salisbury, UK), was cultured in Dulbecco's modified Eagle's medium with GlutaMAX supplemented with 10% (v/v) fetal calf serum (Sigma, St Louis, MO, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. Before treatment, cells were kept at 37 °C, 5% CO2 and 95% humidity. SK-N-SH cells were differentiated with trans-retinoic acid (Sigma) at a final concentration of 10 μM for 5–10 days. In general, to activate the UPR, cells were treated with 0.2 or 0.5 μg/ml TM or with 40 mM 2DG at 37 °C. To inhibit UPR activation, SK-N-SH cells were preincubated for 1 h with 5 mM TUDCA or 0.3 μM PERK inhibitor I, GSK2606414 (PI) (Millipore, Billerica, MA, USA). For the hypothermia experiments, cells were incubated between 26 and 37 °C as indicated. All temperature conditions were normalized and compared with simultaneously incubated 37 °C control condition. To test the reversibility of the UPR, cells were subsequently incubated for 20 h at 37, 26 and 37 °C. The metabolic stressor 2DG was removed by washing the cells two times with regular medium.
5
Investigating Cellular Signaling Pathways
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Trans retinoic acid (RA), LPS, phenylmethanesulfonylfluoride fluoride (PMSF), and niacin were purchased from Sigma (Saint Louis, MO, USA). Compound C (CC) and 3-methyladenine (3-MA) were purchased from Selleck Chem (Shanghai, China).
6
Culturing Murine Microglia and Human Neuroblastoma Cells
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The murine microglial line BV2 was a generous gift from Prof. E. Blasi (Università degli Studi di Modena e Reggio Emilia, Italy); the human neuroblastoma SH-SY5Y line was purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Both lines were cultured in DMEM medium supplemented with 5% fetal calf serum and 100 μM nonessential amino acids, 2 mM L-alanyl-L-glutamine, and 50 mg/ml penicillin-streptomycin (all from Thermo Fisher Scientific, UK) at 37°C in a 5% CO2 atmosphere. SH-SY5Y cells were differentiated to a neuron-like phenotype prior to experimentation by incubation with 10 μM trans-retinoic acid (Sigma-Aldrich, UK) for 5 days [26 (link)].
Primary murine microglia were prepared from C57Bl/6 male mice aged 8 weeks according to our previously published protocols [27 (link)]. Cells were cultured in DMEM medium supplemented with 20% fetal calf serum and 100 μM nonessential amino acids, 2 mM L-alanyl-L-glutamine, and 50 mg/ml penicillin-streptomycin (all Thermo Fisher Scientific, UK) at 37°C in a 5% CO2 atmosphere.
Primary murine microglia were prepared from C57Bl/6 male mice aged 8 weeks according to our previously published protocols [27 (link)]. Cells were cultured in DMEM medium supplemented with 20% fetal calf serum and 100 μM nonessential amino acids, 2 mM L-alanyl-L-glutamine, and 50 mg/ml penicillin-streptomycin (all Thermo Fisher Scientific, UK) at 37°C in a 5% CO2 atmosphere.
7
Cell Culture Conditions for MCF7 and 184B5 Lines
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Cell lines were purchased from the American Type Culture Collection (ATCC, Barcelona, Spain). MCF7 cell lines were grown in Roswell Park Memorial Institute (RPMI) medium (Gibco, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin and 100 mg/mL streptomycin (Gibco, Madrid, Spain). 184B5 cells were grown in DMEM/F12 medium supplemented with 5% FBS, 1x ITS (Lonza, Barcelona, Spain), 100 nM hydrocortisone (Sigma Aldrich, Madrid, Spain), 2 mM sodium pyruvate (Lonza), 20 ng/mL EGF (Sigma Aldrich, Madrid, Spain), 0.3 nM trans-retinoic acid (Sigma-Aldrich) 100 units/mL penicillin and 100 mg/mL streptomycin. Cells were maintained at 37 °C and 5% CO2.
8
Neurite Outgrowth Assay in SH-SY5Y Cells
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Human SH-SY5Y-derived Flp-In host cells19 (link) were cultivated in DMEM-F12 containing 10% FBS, 500 μg/mL of geneticin (G418), and 5 μg/mL of blasticidin. The SH-SY5Y host cells were used to generate and maintain the ΔK280 tauRD-DsRed line as described. SH-SY5Y tauRD-DsRed cells were seeded with all trans-retinoic acid (10 μM; Sigma-Aldrich Co) in six-well (1×105/well) plates.
On the next day, the cells were treated with Congo red (20 μM) or TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 500 μg/mL) for 8 hours. The tauRD-DsRed expression was induced with Dox (1 μg/mL) for 1 week. The cells were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 3% bovine serum albumin, and stained with primary anti-TUBB3 (neuronal class III β-tubulin) antibody (1:1,000; Covance, Princeton, NJ, USA) at 4°C overnight and secondary anti-rabbit Alexa Fluor® 555 antibody (1:500; Molecular Probes, Thermo Fisher Scientific) at room temperature for 3 hours. The total neurite outgrowth of untreated/treated cells was assessed using Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices LLC).
On the next day, the cells were treated with Congo red (20 μM) or TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 500 μg/mL) for 8 hours. The tauRD-DsRed expression was induced with Dox (1 μg/mL) for 1 week. The cells were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 3% bovine serum albumin, and stained with primary anti-TUBB3 (neuronal class III β-tubulin) antibody (1:1,000; Covance, Princeton, NJ, USA) at 4°C overnight and secondary anti-rabbit Alexa Fluor® 555 antibody (1:500; Molecular Probes, Thermo Fisher Scientific) at room temperature for 3 hours. The total neurite outgrowth of untreated/treated cells was assessed using Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices LLC).
9
Cytotoxicity Assays Reagents Purchase
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Trifluoroacetic acid (TFA), propidium iodide (PI), 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyletrazolium bromide (MTT), cis-platinum, taxol, all trans-retinoic acid (ATRA), and 10 hydroxyl-camptothecin (HCPT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cell culture medium RPMI 1640 and fetal bovine serum (FBS) were purchased from GIBCO (Carlsbad, CA, USA). Penicillin-streptomycin and trypsin-versene mixture were purchased from BioWhittaker (Walkersville, MD, USA). Sterilized cell culture materials were purchased from Beckton Dickinson Labware (Franklin Labkes, NJ, USA).
10
Neuroblastoma SH-SY5Y Cell Culture and Immunoprecipitation
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Human neuroblastoma SH-SY5Y cells (American Type Culture Collection #CRL-2266).
Trypsin (Promega, Sequencing grade, #V5113) stored at −80 °C.
Dichlorvos CAS 62-73-7 (Chem Service Inc. #N11675) 0.01 M stock solution in acetonitrile stored at −80 °C.
DMEM/F12 GlutaMAX (Gibco #10565–108).
Fetal bovine serum (Life Tech #16000044).
Penicillin & streptomycin (Gibco #15140–122).
Trans retinoic acid (Sigma-Aldrich #554720).
Pierce IP lysis buffer (25 mM TrisCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) (Thermo Scientific #87787).
Halt protease inhibitor cocktail (Thermo Fisher Scientific #78430); containing AEBSF, aprotinin, bestatin, E–64, leupeptin and pepstatin A.
RIPA buffer (25 mM Tris-HCl pH 7.6, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) (Thermo Fisher Scientific #89900).
Mouse anti-isopeptide monoclonal 81D1C2 (LS Bio #LS-C153331); reconstituted with water to 1 mg/mL.
Protein G agarose (Protein Mods LLC #PGGH); binds 20 mg antibody per ml beads.
Bicinchoninic acid protein assay kit (Thermo Scientific #23228).
Trypsin (Promega, Sequencing grade, #V5113) stored at −80 °C.
Dichlorvos CAS 62-73-7 (Chem Service Inc. #N11675) 0.01 M stock solution in acetonitrile stored at −80 °C.
DMEM/F12 GlutaMAX (Gibco #10565–108).
Fetal bovine serum (Life Tech #16000044).
Penicillin & streptomycin (Gibco #15140–122).
Trans retinoic acid (Sigma-Aldrich #554720).
Pierce IP lysis buffer (25 mM TrisCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) (Thermo Scientific #87787).
Halt protease inhibitor cocktail (Thermo Fisher Scientific #78430); containing AEBSF, aprotinin, bestatin, E–64, leupeptin and pepstatin A.
RIPA buffer (25 mM Tris-HCl pH 7.6, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) (Thermo Fisher Scientific #89900).
Mouse anti-isopeptide monoclonal 81D1C2 (LS Bio #LS-C153331); reconstituted with water to 1 mg/mL.
Protein G agarose (Protein Mods LLC #PGGH); binds 20 mg antibody per ml beads.
Bicinchoninic acid protein assay kit (Thermo Scientific #23228).
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