Human liver cell line (THLE-3), cancer cell lines (SNU-475 and SNU-423), normal B lymphocyte (CCL-156) and normal monocyte/macrophage (CRL-9855) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). SNU-475, SNU-423 and CCL-156 were cultured in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO) and 1% penicillin and streptomycin. THLE-3 was grown in Bronchial Epithelial Cell Growth Medium (BGEM) bulletkitTM (Lonza/Clonetics Corporation, Walksrsville, MD 21793) whereas, CRL-9855 was grown in Iscove’s Modified Dulbecco’s Medium (IMDM, ATCC 30-2005, Manassas, Virginia, USA) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.05 mM 2-mercaptoethanol, 0.1 mM hypoxanthine and 0.016 mM thymidine, 90%; fetal bovine serum, 10%. All cell lines were cultured in a humidified incubator with 5% CO2 at 37 °C. All experiments were conducted on cell lines with passage number 1 to 10.
Crl 9855
Manufactured by American Type Culture Collection
Sourced in United States, Germany
The CRL-9855 is a laboratory equipment product offered by American Type Culture Collection. It is designed for cell culture applications. The core function of the CRL-9855 is to provide a controlled environment for the growth and maintenance of cells.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Merck Group (2 mentions)
Snu 423, by Merck Group (1 mentions)
Snu 475, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Thle 3, by American Type Culture Collection (1 mentions)
Snu423, by American Type Culture Collection (1 mentions)
Snu475, by American Type Culture Collection (1 mentions)
Thle 3, by Lonza (1 mentions)
Bgem bulletkittm, by Lonza (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Mycoplasma, by Promega (1 mentions)
Multi well culture plates, by Corning (1 mentions)
Fluospheres carboxylate modified microsphere, by Thermo Fisher Scientific (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Facscelesta cell analyzer, by BD (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
6 protocols using crl 9855
1
Cell Culture Protocols for Diverse Cell Lines
2
Monocyte-to-Macrophage Differentiation Protocol
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Undifferentiated human monocytes (CRL-9855™) were purchased from ATCC® and sub-cultured in RPMI 1640 (Merck, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% sodium pyruvate (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2.
For differentiation into macrophages, the monocytes were seeded in 96 multi-well culture plates (0.5 × 104 cells/well) for the MTT assay and in 150 mm cell culture dishes (0.1 × 107 cells/dish) for the immunoblotting and stimulated with 100 ng/mL of PMA (phorbol-12-myristate-13-acetate, purchased from Merck, Darmstadt, Germany, stock solution 1 mM in DMSO) in complete RPMI for 48 h at 37 °C and 5% CO2. After 48 h, a culture of macrophage-like cells was obtained and used for further experimental procedures.
For differentiation into macrophages, the monocytes were seeded in 96 multi-well culture plates (0.5 × 104 cells/well) for the MTT assay and in 150 mm cell culture dishes (0.1 × 107 cells/dish) for the immunoblotting and stimulated with 100 ng/mL of PMA (phorbol-12-myristate-13-acetate, purchased from Merck, Darmstadt, Germany, stock solution 1 mM in DMSO) in complete RPMI for 48 h at 37 °C and 5% CO2. After 48 h, a culture of macrophage-like cells was obtained and used for further experimental procedures.
3
Human Monocyte and Dental Pulp Cell Culture
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Human monocytes (CRL-9855™) were purchased from ATCC® and sub-cultured in RPMI 1640 (EuroClone, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% sodium pyruvate (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2.
Primary human dental pulp cells (HDPCs) were obtained as previously reported [33 (link)]. According to the Italian Legislation and the code of ethical principles for medical research involving human subjects of the World Medical Association (Declaration of Helsinki), young donors who underwent extraction of the third molar signed an informed consent form. This project has received the approval of the Local Ethical Committee of the University of Chieti-Pescara (approval number 1173, date of approval 31 March 2016). HPCs were maintained in MEM alpha (EuroClone, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA).
Primary human dental pulp cells (HDPCs) were obtained as previously reported [33 (link)]. According to the Italian Legislation and the code of ethical principles for medical research involving human subjects of the World Medical Association (Declaration of Helsinki), young donors who underwent extraction of the third molar signed an informed consent form. This project has received the approval of the Local Ethical Committee of the University of Chieti-Pescara (approval number 1173, date of approval 31 March 2016). HPCs were maintained in MEM alpha (EuroClone, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA).
4
Cell Culture and Treatment Conditions
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THP1 cells were cultured in RPMI1460 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 μg/mL streptomycin and 100 U/ml penicillin at 37°C in 5% CO2. MCF7, BT549 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 μg/mL streptomycin and 100 U/ml penicillin at 37°C in 5% CO2. Cells were treated with indicated concentrations of therapies for indicated periods of time. All cell lines and monocytes (CRL9855) were obtained from ATCC (Sykes et al., 2016 (link)). Cell lines were tested for Mycoplasma (Promega) and authenticated by the ATCC Cell Authentication Testing Service (Lee et al., 2020 (link)).
5
Monocyte to Macrophage Differentiation
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Undifferentiated human monocytes (CRL-9855™) were purchased from ATCC® and sub-cultured in RPMI 1640 (Merck, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% sodium pyruvate (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2. Differentiated macrophages were obtained as previously described26 (link). Both the cell types were seeded at 0.5 × 105 cells/well in multi-well culture plates (Corning, Falcon®, Glendale, Arizona, USA). To establish an inflamed environment, cells were stimulated with LPS 0.5 µg/mL (lipopolysaccharide from Escherichia coli, purchased from Merck, Darmstadt, Germany, stock solution 1 mg/mL in water). Untreated and LPS-stimulated samples were harvested immediately after treatment (T0 = time zero) and after 3 and 24 h and used for further procedures.
6
Differentiation and Characterization of SC Macrophages
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The SC cell is a non-cancerous human monocytic cell line that can be differentiated into resting macrophages (SC-macrophages, SCM) [36] (link). To prepare SCM, 2 × 105 SC cells (CRL-9855, ATCC, Virginia, USA) per well were plated in 6-well plates and cultured with complete RPMI supplemented with 10% FBS at 37 °C, 5% CO2. SC cells were exposed to 200 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 3 d to allow monocytic differentiation, followed by the removal of PMA and continued 5-day culture to obtain resting SCM.
Flow cytometry (FCM) was performed to measure the CD14 expression on SC cells and SCM. Cells were washed twice with PBS, and treated with Human Trustain FcX (BioLegend, Beijing, China) for 15 min to block the Fc receptors. Cells were then stained with FITC conjugated anti-human CD14 antibody or anti-mouse IgG isotype control antibody (Biolegend) for 20 min in dark. For phagocytosis analysis, cells were treated with 2 mL 0.5 µm FluoSpheres carboxylate-modified microspheres (505/515, 1.68 × 108 particles/mL, Thermo). After PBS washes, cells were resuspended and filtered by 70 µm sieve meshes. A FACSCelesta™ Cell Analyzer (BD Biosciences, Shanghai, China) was used for all FCM analyses.
Flow cytometry (FCM) was performed to measure the CD14 expression on SC cells and SCM. Cells were washed twice with PBS, and treated with Human Trustain FcX (BioLegend, Beijing, China) for 15 min to block the Fc receptors. Cells were then stained with FITC conjugated anti-human CD14 antibody or anti-mouse IgG isotype control antibody (Biolegend) for 20 min in dark. For phagocytosis analysis, cells were treated with 2 mL 0.5 µm FluoSpheres carboxylate-modified microspheres (505/515, 1.68 × 108 particles/mL, Thermo). After PBS washes, cells were resuspended and filtered by 70 µm sieve meshes. A FACSCelesta™ Cell Analyzer (BD Biosciences, Shanghai, China) was used for all FCM analyses.
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