REF52 (an immortal line of postcrisis Fischer rat embryo cells)4 (link)62 (link)63 (link) and NIH3T3 mouse fibroblasts (CRL-1658, ATCC) were routinely grown in Minimum Essential Medium Alpha Medium (Gibco/Invitrogen) supplemented with 10% bovine growth medium (BGS, Hyclone/Thermo Scientific). For the preparation of E2F dynamics measurement by using time-lapse microscopy, cells were first trypsinized, resuspended at a density of 1 × 105 per ml and then seeded in μ-Slide I (tissue culture treated, ibidi) channel slides by adding 1 ml volume of the cell suspension. After overnight culturing, cells were synchronized in quiescence by shifting into Minimum Essential Medium Alpha medium with 0.02% BGS (starvation medium) for 36 h. For perturbation experiments, PD0332991 (CDK4/6 inhibitor, ChemieTek), CVT-313 (CDK2 inhibitor, Enzo Life Science), 10058-F4 (c-Myc inhibitor, Sigma Aldrich) and (+)-JQ1 (bromodomain and extra terminal domain inhibitor, Cayman Chemical) were added into cells immediately after cells were released from serum starvation (by adding 10% BGS) in either single or combined way. For 92 h perturbation experiment with both PD0332991 and CVT-313, cells were growing with replaced fresh medium with fresh inhibitors after the initial 48 h.
Crl 1658
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom
CRL-1658 is a cell line from the American Type Culture Collection. It is derived from human cervical cancer cells. This cell line is routinely used in cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Nih3t3, by American Type Culture Collection (7 mentions)
Tib 71, by American Type Culture Collection (3 mentions)
Calf serum, by Biosera (3 mentions)
Infinite m200 pro nanoquant absorbance reader, by Tecan (3 mentions)
Dulbecco s modified eagle s medium, by Biosera (3 mentions)
Penicillin streptomycin, by GE Healthcare (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Ccl 153, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Ccl 2, by American Type Culture Collection (2 mentions)
Crl 3216, by American Type Culture Collection (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Neuro 2a, by American Type Culture Collection (2 mentions)
70 protocols using crl 1658
1
E2F Dynamics Measurement in Synchronized Cells
2
Culturing Human and Animal Cell Lines
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Cultures of Human foreskin fibroblast (HFF; HFF-1 ATCC® SCRC-1041™), Madin–Darby bovine kidney (MDBK; ECACC) and mouse embryonic fibroblast (NIH/3T3; ATCC® CRL-1658™) cells were retrieved from -80°C stock and cultured continuously at 37°C under atmosphere 5% CO2 in our laboratory. The NIH/3T3 and HFF cell lines were maintained in Dulbecco Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA), while MDBK cell line grown in Minimum Essential Medium Eagle (MEM; Gibco) and cell cultivation was performed as describe everywhere [3 (link), 4 (link)].
3
Cytotoxicity Assessment of Encapsulated EPPR
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The MTT cytotoxicity assay was performed as described previously by Tsuboy et al. [51 (link)]. For this assay, mouse fibroblasts of dermal origin (NIH/3T3, ATCC® CRL-1658™) were inoculated into 96-well microtiter plates and incubated in culture medium for 24 h at 37 °C under 5% carbon dioxide (CO2). After reaching approximately 75% confluence (24 h), these cells were exposed to five different concentrations of the encapsulated EPPR (31.25, 62.5, 125, 250, and 500 μg/mL). In addition, to understand the cytotoxicity of the encapsulated EPPR, acetic acid was evaluated at the same proportions used in the encapsulation process (0.015%, 0.030%, 0.060%, 0.120%, and 0.240%). For the NC, the extract was replaced with a physiological solution, and for the PC, it was replaced with 2% (v/v) Tween 80. The treatment times were 24, 48, and 72 h.
EPPR was not included in this screening because Pegorin Brasil et al. [10 (link)] analyzed the same extract and showed that it did not present significant cytotoxicity.
EPPR was not included in this screening because Pegorin Brasil et al. [10 (link)] analyzed the same extract and showed that it did not present significant cytotoxicity.
4
Cytotoxicity of T. toxicaria Extracts
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The cytotoxicity of T. toxicaria extracts (0.001, 0.01, 0.1 and 1.0 mg/mL) was evaluated using Mosmann’s [40 (link)] method. Hepatocellular carcinoma cells (HepG2, ATCC® HB8065™) and murine fibroblast cells (3T3, ATCC® CRL-1658™) were donated by Dr. Viviane Souza do Amaral and Dr. Silvia Regina Batistuzzo de Medeiros, respectively, from Department of Cellular Biology and Genetics, UFRN, Natal-RN, Brazil. HepG2 and 3T3 cells (5 x 104 cells) were cultivated (Incubator HEPA Filter Model 3110 Themoforma Serie II Water CO2, USA) in Dulbecco’s modified Eagle (DMEM; Cultilab, Brazil) and supplemented with 10% fetal bovine serum (FBS; Cultilab, Brazil), 2% L-glutamine (Sigma-Aldrich, USA) and 1% streptomycin/penicillin (Sigma-Aldrich, USA), at 37°C and 5% CO2. The control included cells cultivated in DMEM and 10% FBS, and was able to promote 100% reduction of MTT (1.0 mg/mL), considered as 100% of cell proliferation. Results were expressed as the percentage of MTT reduction, using the following equation: MTT Reduction (%) = (Ae–Ac) x 100, where Ae: absorbance of cells subjected to treatment with extracts, and Ac: absorbance of control.
5
Cytotoxicity Assay using NIH/3T3 Cells
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The NIH/3T3 mouse embryonic fibroblast cell line (ATCC® CRL-1658™, London, UK) was used for cytotoxicity assays. The incubation period of NIH/3T3 cells was based on the supplier's recommendation. NIH/3T3 cells were seeded at 1 × 104 cells into each well of 96-well plates. MTT assay was carried out in accordance with the standards previously described.19,21 (link)
6
Mouse Cell Line Culture and Treatments
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In this experimental study, NIH3T3 mouse
embryonic fibroblasts (CRL1658, ATCC, USA) and
AML12 mouse hepatic cells (CRL-2254™, ATCC)
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen, USA) supplemented with 10%
fetal bovine serum (Sigma-Aldrich, USA) at 37˚C in
a humidified atmosphere of 5% CO2. The cells were
passaged every two days at 1:6 ratios. The cells were
treated with 500 nM Thapsigargin (Sigma-Aldrich,
USA) or 100 ng/ml Tunicamycin (Sigma-Aldrich,
USA) unless otherwise noted. Human lung cancer cell
line H1299 (CRL5803, ATCC), human ovarian cancer
line SKOV3 (HTB-77, ATCC), human ovarian surface
epithelial cell line HOSE (Beinachuanglian, PRC) and
human lung fibroblast HFL1 (CCL153, ATCC) cells
were cultured in RPMI 1640 (Invitrogen, USA) with
10% fetal bovine serum.
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In this experimental study, NIH3T3 mouse
embryonic fibroblasts (CRL1658, ATCC, USA) and
AML12 mouse hepatic cells (CRL-2254™, ATCC)
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen, USA) supplemented with 10%
fetal bovine serum (Sigma-Aldrich, USA) at 37˚C in
a humidified atmosphere of 5% CO2. The cells were
passaged every two days at 1:6 ratios. The cells were
treated with 500 nM Thapsigargin (Sigma-Aldrich,
USA) or 100 ng/ml Tunicamycin (Sigma-Aldrich,
USA) unless otherwise noted. Human lung cancer cell
line H1299 (CRL5803, ATCC), human ovarian cancer
line SKOV3 (HTB-77, ATCC), human ovarian surface
epithelial cell line HOSE (Beinachuanglian, PRC) and
human lung fibroblast HFL1 (CCL153, ATCC) cells
were cultured in RPMI 1640 (Invitrogen, USA) with
10% fetal bovine serum.
7
Cell Culture Conditions for Fibroblasts
8
Generation of LMNA Mutation Cell Line
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NIH/3T3 cells (ATCC, CRL-1658TM) were used for single guide RNA (sgRNA) optimization. Mouse embryonic fibroblasts (MEFs) (ATCC, SCRC-1008TM) were used as feeders for mouse embryonic stem (mES) cells. R1/E mES cells (ATCC, SCRC-1036TM) were used as wild type (WT) control and for the generation of a patient-specific knock in the LMNA mutation cell line. All cells were handled and cultured according to the protocols provided by ATCC. MEFs were mitotically arrested by irradiation.
9
Murine Cell Lines for Research
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The following murine cell lines were used in this study: brain endothelial cells (bEnd.3), pancreatic islet endothelial cells (MS1), heart endothelial cells (H5V), and fibroblast cells (NIH 3T3). bEnd.3 and H5V cell lines were generous gifts from prof. Enrico Mastrobattista (Department of Pharmaceutical Sciences, Utrecht, The Netherlands) and prof. Ingrid Molema (Department of Pharmacology, Groningen, The Netherlands), respectively. MS1 (ATCC® CRL-2279TM) and NIH 3T3 were (ATCC® CRL-1658™) purchased from ATCC. All cell lines except MS1 were cultured in Dulbecco’s modified eagle’s medium (Lonza, Basel, Switzerland) containing 4.5 g/L glucose and glutamine supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 10% fetal bovine serum (FBS, Sigma-Aldrich). MS1 cells were grown in DMEM from ATCC (30-2002™). Oral squamous cell carcinoma cell line OSC-19-luc2-cGFP (OSC, Radiology and Molecular Imaging department, Leiden, The Netherlands) were cultured as described previously [19 (link)].
10
Cell Culture Conditions Optimization
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