The active metabolite of vitamin D3, 1,25(OH)2D3, and the inactive preform, 25(OH)D3, were purchased from Tocris Bioscience (Bristol, UK). Purified lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Heat-killed pneumococcal bacteria serotype 19F (HK19F; reference strain originally obtained from the University of Alabama) was prepared by harvesting the mid-log phase of the bacterial culture and incubating in a water bath at 80 °C for 60 min. The bacterial concentration was determined before and after heat-killing by plating on horse blood agar plates. The HK19F was stored in aliquots at −80 °C prior to use, and the same batch was used throughout the study. Neutrophils were derived by stimulation of a HL-60 cell line (ATCC® CCL-240™, Manassas, VA, USA) with 100 mM N,N-dimethylformamide for five days.
Ccl 240
Manufactured by American Type Culture Collection
Sourced in United States, Germany
The CCL-240 is a cell culture incubator designed for the maintenance and growth of cell lines. It provides a controlled environment with adjustable temperature, humidity, and CO2 levels to support optimal cell culture conditions.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Crl 1780, by American Type Culture Collection (4 mentions)
Tib 202, by American Type Culture Collection (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Famse, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hl 60, by American Type Culture Collection (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Htb 22, by American Type Culture Collection (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
Crl 11268, by American Type Culture Collection (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
Htb 96, by American Type Culture Collection (1 mentions)
Penicillin, by Duchefa Biochemie (1 mentions)
36 protocols using ccl 240
1
Vitamin D Metabolites in Neutrophil Activation
2
Routine Cell Culture Protocols
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Cell lines were purchased at ATCC (HEK293T: ATCC® CRL-11268™, U2OS: ATCC® HTB-96™, HL-60: ATCC® CCL-240™) and were tested on regular basis for mycoplasma contamination. Cultures were discarded after 2–3 months of use. HEK293T (human embryonic kidney) and U2OS (human osteosarcoma) cells were cultured at 37 °C under 7% CO2 in DMEM containing phenol red, stable glutamine, 10% (v/v) high iron newborn calf serum (Seradigm), penicillin and streptomycin (200 μg/mL each; Duchefa). Medium was refreshed every 2–3 days and cells were passaged two times a week at 80–90% confluence. HL-60 (human promyeloblast) cells were cultured at 37 °C under 5% CO2 in HEPES-supplemented RPMI containing phenol red, stable glutamine, 10% (v/v) fetal calf serum (Biowest), penicillin and streptomycin (200 μg/mL each), unless stated otherwise. Cell density was maintained between 0.2 × 106 and 2.0 × 106 cells/mL. Cell viability was assessed by Trypan Blue exclusion and quantification using a TC20™ Automated Cell Counter (Bio-Rad).
3
Cultivation of Hematological Cell Lines
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The THP-1 (TIB-202, ATCC, Manassas, VA, USA), U937 (CRL-1593.2, ATCC), HL-60 (CCL-240, ATCC), MOLM13 (C6600, Beyotime, Shanghai, China), K562 (CCL-243, ATCC), and Jurkat (CRL-2898, ATCC) cell lines were cultivated in RPMI 1640 (11875093, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, SH30084, Hyclone, Logan, UT, USA), 1% penicillin–streptomycin (SV30010, Hyclone), 1% HEPES (H3375-25G, Sigma-Aldrich, St. Louis, MO, USA) and 2 mM glutamine (25-005-CI, Corning, Salt Lake City, UT, USA). All cells were maintained in a humidified incubator at 37 °C with 5% CO2.
4
Evaluating AshwaSR's Inhibitory Effect on Superoxide Production
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The inhibition of superoxide production by AshwaSR was evaluated in phorbol 12- myristate 13- acetate (PMA)-induced HL-60 (ATCC, Cat# CCL-240, Manassas, VA, USA) human monocytic cells. These HL-60 cells were washed with Hank’s Balanced Salt Solution (HBSS) once and were then taken into 50 mL falcon tubes. Lucigenin (working concentration 200μM) was added to the falcon tube and 50000 cells/well were added in a 96-well white plate (Corning, Cat# 3917, Somerville, MA, USA). The cells were immediately stimulated with 100nM PMA, either in the absence or presence of varying concentrations (1, 2.5, 5, 7.5, and 10 µg/mL) of AshwaSR or 1, 2.5, 5, 10 and 20 µg/mL of the positive control (green tea extract, obtained from the internal phytochemistry department). A spectrophotometer (Enspire Multimode Reader, Perkin Elmer) was set in the kinetic mode and the relative luminescence was measured up to 1 hour.
5
Cell Culture Protocols for Cancer and Non-Cancer Cell Lines
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HL-60 and HL-60R cells were cultured in Roswell Park Memorial Institute (RPMI) 1640. hTERT RPE-1 and 1-7HB2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone Europe Ltd., Cramlington, UK), only for 1-7HB2 cells, supplemented with hydrocortisone (5 μg/mL) and insulin (10 μg/mL). All media were supplemented with 10% heat inactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin (all reagents were from HyClone Europe Ltd., Cramling-ton, UK) in a humidified atmosphere at 37 °C in 5% CO2. HL-60 cells were obtained from ATCC® (CCL-240, Rockville, MD, USA), while its variant HL-60R was selected for multidrug resistance (MDR) by exposure to gradually increasing concentrations of doxorubicin. The hTERT RPE-1 (ATCC® CRL-4000TM) cells were kindly provided by Patrizia Cancemi (Department of Biological, Chemical and Pharmaceutical Science and Technology, University of Palermo, Italy). The 1-7HB2 (ECACC 10081201-Cancer Research Technology, London, UK) cells were kindly provided by Giulio Ghersi (Department of Biological, Chemical and Pharmaceutical Science and Technology, University of Palermo, Palermo, Italy).
6
HL-60 Cell Differentiation Protocol
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The human promyelocytic leukemia HL-60 cell line (33 (link)) was purchased from ATCC® (CCL-240™). The culture medium used was RPMI-1640 (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 10% v/v complement heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 µg/mL streptomycin (Life Technologies) and 100 U/mL of penicillin (Life Technologies). Cells were cultured at 37°C in a humidified atmosphere with 5% CO2 and were passaged at a density of 1x106/mL. Differentiation towards neutrophil-like cells was performed in fresh complete RPMI 1640 containing DMSO (1.3% v/v) for 4.5 days (34 (link)).
7
Establishment and Characterization of HNSCC and AML Cell Lines
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Five human HNSCC cell lines were used: SCC040 (German Culture Collection, DSMZ (#ACC660)), FaDu (ATCC (HTB-43)), VU40T (Prof H. Joenje (VU University Medical Centre, Amsterdam)), HN12 (Dr J.F. Ensley (Wayne State University, Detroit, MI)) and UDSCC2 (Dr Henning Bier (University of Duesseldorf, Germany)). The cell lines were authenticated using STR profiling (NorthGene, UK). HNSCC cell lines were maintained in DMEM (Sigma- Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin-streptomycin (Gibco), 4 mM l -glutamine (Sigma-Aldrich), 1X non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Primary human oral keratinocyte (HOK) cells were purchased from Caltag Medsystems and cultured over Poly-l -Lysine in Oral Keratinocyte Medium supplemented with 1% oral keratinocyte growth supplement (all from ScienceCell) and 1% penicillin-streptomycin. AML cell lines (SKM-1 (from Dr Stefan Heinrichs, University of Essen, Germany) and HL-60 (ATCC, CCL-240)) were cultured in RPMI1640 medium (ThermoFisher Scientific), supplemented with 15% FBS (Sigma-Aldrich), 1% l -glutamine and 1% penicillin-streptomycin. All cell lines were regularly tested for mycoplasma using MycoAlert (Lonza).
8
Evaluating EOs and HYs on Cell Viability
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To evaluate the effect of EOs and HYs on cell viability, five different cell lines were used: human acute promyelocytic leukemia cells (HL60, ATCC® CCL-240), human neuroblastoma cells (SH-SY5Y, ATCC® CRL-2266), human metastatic adenocarcinoma breast cells (MCF7, ATCC® HTB-22™), human adenocarcinoma breast cells (MDA, ATCC® MB-231), and normal breast epithelial cell (MCF10A. ATCC® CRL-10317™).
The HL60 were cultured in RPMI-1640 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The SH-SY5Y, MCF7, and MDA cells were cultured in DMEM F-12 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The MCF10A were maintained in DMEM F-12 medium supplemented with 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% Horse Serum (HS), 1% glutamine, and 1% penicillin-streptomycin; before being seeded for the viability assay, the culture medium was deprived of EGF and the HS was reduced to 2%. All the cell lines were maintained at 37 °C in a humidified 5% CO2 condition. All experiments were performed with the cells in their logarithmic growth phase.
The HL60 were cultured in RPMI-1640 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The SH-SY5Y, MCF7, and MDA cells were cultured in DMEM F-12 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The MCF10A were maintained in DMEM F-12 medium supplemented with 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% Horse Serum (HS), 1% glutamine, and 1% penicillin-streptomycin; before being seeded for the viability assay, the culture medium was deprived of EGF and the HS was reduced to 2%. All the cell lines were maintained at 37 °C in a humidified 5% CO2 condition. All experiments were performed with the cells in their logarithmic growth phase.
9
Phagocytosis of S. pneumoniae by Human Granulocytes
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Experiments investigating phagocytosis were performed using a flow cytometry assay including S. pneumoniae labeled with 5, 6-carboxyfluorescein succinimidyl ester (FAM-SE; Molecular Probes) and human HL-60 cells (CCL-240; ATCC) differentiated to granulocytes [14 (link),15 (link),27 (link),28 (link)]. Infection assays were performed with a ratio of 10 bacteria per cell. Results were expressed as a RFI defined as the proportion of positive cells for fluorescent bacteria multiplied by the geometric mean of fluorescence intensity, which correlates with the amount of bacteria phagocytosed per cell [26 (link),27 (link)].
10
Differentiation of HL-60 Cells for Macrophage Assays
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Digestates from RRS and RCS aqueous extracts were incorporated in the in vitro model of macrophage-like cells obtained by the differentiation of the human acute myeloid leukemia cell line HL-60 (ATCC® CCL-240™) as previously described. 27 Briefly, the cells were kept undifferentiated and growing at an exponential ratio in complete culture media (CCM), RPMI-1640 (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (GIBCO Invitrogen, Paisley, UK) and 1%
penicillin/streptomycin (GIBCO) prior to their differentiation. Growing conditions were set at 37 °C and under a 5% CO 2 atmosphere in the incubator. Cells were allowed to differentiate for conduction of the in vitro assays, always between passages number 5 and 20.
penicillin/streptomycin (GIBCO) prior to their differentiation. Growing conditions were set at 37 °C and under a 5% CO 2 atmosphere in the incubator. Cells were allowed to differentiate for conduction of the in vitro assays, always between passages number 5 and 20.
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