Monomorphonuclear cells’ isolation was performed with the methods described above. Cells were suspended in culture medium: Gibco AIM-V serum-free (Life Technologies, USA). Unstimulated cells served as a control culture. Other cultures were stimulated with α-GalCer (Enzo Life Sciences, USA) and cytokines (BioVision Inc., USA) in the following combinations: I: α-GalCer+IL-2, II: α-GalCer+IL-2+IL-7, III: α-GalCer+IL-2+IFN-γ. Cells were cultured at 37°C, 5% CO2 and 95% humidity in a New Brunswick Galaxy 170R incubator. Seven days after culture initiation, their medium was replaced and the same set of stimulators was added. After that cells were incubated for another 7 days. On the last day of culture, the percentage of iNKT+CD3+ cells was assessed by the method described above.
Galaxy 170r incubator
Manufactured by Eppendorf
Sourced in United States
The Galaxy 170R is a high-quality incubator designed for laboratory use. It offers a temperature range of 4°C to 60°C and can maintain precise temperature control. The incubator provides a reliable and consistent environment for various cell culture and sample incubation applications.
Automatically generated - may contain errors
Lab products found in correlation
Humec ready medium, by Thermo Fisher Scientific (2 mentions)
α galcer, by Enzo Life Sciences (1 mentions)
α galcer, by Abcam (1 mentions)
Mg 63 cells, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Gentlemacs dissociator and mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Falcon flasks, by BD (1 mentions)
Luna 2 automated cell counter, by Logos Biosystems (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
U118mg, by American Type Culture Collection (1 mentions)
Glutamine, by Biosera (1 mentions)
Caco 2 cells, by European Collection of Cell Cultures (1 mentions)
Huvec tert 2, by American Type Culture Collection (1 mentions)
Endothelial cell growth medium 2, by Lonza (1 mentions)
Mir 9 5p mimic, by GenePharma (1 mentions)
Lipofectamine 2000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
10 protocols using galaxy 170r incubator
1
Isolation and Stimulation of iNKT Cells
2
Inhibitor Effects on C. burnetii Growth
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Growth curves. Vented flasks containing 5 mL of ACCM-2 media supplemented with 0.50 mM L-tryptophan and containing either 100 μM of SF235 or AN296 or DMSO vehicle control were inoculated at 1 × 104 CFU/mL using freshly thawed stocks of C. burnetii NMI. Cultures were incubated statically at 37°C in a Galaxy 170 R incubator (New Brunswick Scientific) adjusted to 5 % CO2 and 2.5 % O2. Samples of 100 μL were removed at days 0, 1, 2, 3, 4 and 7 and plated in serial dilutions on ACCM-2 agar plates which were then left for 7–10 days before viability counts were performed. For delayed dosing experiments, the assay was prepared without inhibitors as described above and on day 3 of the growth curve, 25 μL of inhibitor or DMSO diluted in media was added to each culture.
3
Evaluating Biocompatibility of Gelatin Nanoparticles
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In order to show that the GNPs were biocompatible with living cells and that after purification no cytotoxic residues such as acetone or GTA from the production steps were present, a cell culture experiment was carried out. MG-63 cells (ATCC CRL 1427) were used. For each sample 50,000 cells were placed in a 12-well plate on glass plates, which were previously coated with polylysine. The cells were cultivated overnight with Dulbecco’s Modified Eagle Medium (DMEM F12 containing F12 nutrient and the additions of 1% penicillin/streptomycin (P/S, Sigma Aldrich (now Merck), Darmstadt, Germany) and 10% fetal bovine serum (FBS, Merck, Darmstadt, Germany) in a New Brunswick Galaxy 170 R incubator (Eppendorf, Hamburg, Germany) at 37 °C and a CO2 saturation of 5%. The next day, a production batch (from originally 1.25 g gelatin) GNP were washed in 1 mL ethanol (60%) to prevent cell culture contamination. The GNP were dissolved in the culture medium and pipetted to the cells. Nunc™ Thermanox™ Coverslip (Thermo Fisher Scientific, Waltham, MA, USA) membranes were used as a control. For each time point at least three samples were used and all experiments were repeated at least three times.
4
Dissociation and culture of primary tumor cells
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Primary epithelial cells (from three distinct tumors (namely #1, #2, #3) from each mouse model or from three PDX implants) were obtained using the gentle MACS™ Dissociator and Mouse Tumor dissociation kit (Miltenyi Biotec, Bisley, Surrey, UK) following the manufacturer’s recommendations using the protocol for ‘Dissociation of Tough Tumors’ for mouse tumors and the protocol for ‘Dissociation of Soft and Medium Tumors’ for the PDX. To ensure efficient dissociation volumes of Enzyme D, Enzyme R and Enzyme A were scaled up according to the size of the tumor piece (100 μL, 50 μL and 12.5 μL respectively per each 0.5 cm3). The optional steps - the short spin for collection of the dissociated material at the bottom of the MACS tube and red blood cell lysis - were included in the procedure.
Mouse cells were cultured in complete growth medium in 2D adherent conditions for expansion or in 3D for functional studies. Cells up to passage 5 were used for all the experiments in this study. Freshly isolated human PDX cells were grown in HuMEC Ready Medium (Thermo Fisher Scientific) in Matrigel in 3D. Cultures were maintained at 37°C in a 5% CO2/5%O2 atmosphere in a Galaxy 170R incubator (New Brunswick, Eppendorf).
Mouse cells were cultured in complete growth medium in 2D adherent conditions for expansion or in 3D for functional studies. Cells up to passage 5 were used for all the experiments in this study. Freshly isolated human PDX cells were grown in HuMEC Ready Medium (Thermo Fisher Scientific) in Matrigel in 3D. Cultures were maintained at 37°C in a 5% CO2/5%O2 atmosphere in a Galaxy 170R incubator (New Brunswick, Eppendorf).
5
Dissociation and Culture of Primary Tumour Cells
6
Glioblastoma and Colorectal Cancer Cell Cultivation
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Human glioblastoma cell line U118MG, and human colorectal adenocarcinoma cell line DLD-1 were provided by American Type Culture Collection (ATCC). Cells were maintained in high glucose DMEM with addition of 10% of heat-inactivated fetal bovine serum Gold (FBS Gold), penicillin (100 U/mL), streptomycin (100 μg/mL), and 2 mmol/mL L-glutamine. The cells were cultured in Falcon flasks (BD Pharmingen™, San Diego, CA, USA) in Galaxy 170R incubator (Eppendorf Inc., Hamburg, Germany) in a humidified atmosphere of 5% CO2 in the air, at 37 °C. Cells reaching sub-confluency were detached from the culture dishes using 0.05% trypsin, 0.02% EDTA in calcium-free phosphate-buffered saline (PBS) and counted in Luna-II™ automated cell counter (Logos Biosystems, Annandale, VA, USA). CA was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted in growth medium keeping the final concentration of DMSO ≤ 0.5% in cultures.
7
Maintaining B. burgdorferi Strains for Research
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An infectious, low-passage (<5 passages) B. burgdorferi strain B31 A3, as well as isogenic uvrB and mutS1-deficient strains were maintained at 34°C in BSK II medium, pH 7.6 under microaerobic conditions (3% O2, 5% CO2) in a Galaxy 170R incubator (Eppendorf, Hauppauge, NY, USA) as previously described (Bourret et al., 2011 (link); Troxell et al., 2014 (link)). Cell density was determined by dark field microscopy. The plasmid profiles of each strain were confirmed by PCR (Bunikis et al., 2011 (link)). Construction of additional BER, MMR, and NER mutant strains is described in the Supplementary Material using primers listed in Supplementary Table S2 and strains are listed in Supplementary Table S1 .
8
Cultivation and Characterization of Caco-2 and HUVEC/TERT 2 Cells
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Human White colon adenocarcinoma (Caco-2) cells (European Collection of Cell Cultures (ECACC, UK) were isolated from a primary colonic tumor in a 72-year-old White male using the explant culture technique. During routine subculture, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with the supplementation of 10% fetal bovine serum (FBS), 1% non-essential amino acid and penicillin–streptomycin solution, at 37 °C, in an incubator containing 5% CO2. The passage number of Caco-2 cells was 25–40 in the study.
HUVEC/TERT 2 was obtained from ATCC (ATCC, Manassas, VA, USA). The cell line was isolated from the vascular endothelium of a White female patient. Cells were maintained in M199 with the supplementation of 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% amphotericin B, 2 mM glutamine (Biosera, Nuaille, France), and Endothelial Cell Growth Medium-2 (Lonza, Basel, Switzerland), at 37 °C, in a Galaxy 170R incubator under 5% CO2 (Eppendorf, Hamburg, Germany). Adhesion of the cells was support with a 0.1% gelatin solution.
HUVEC/TERT 2 was obtained from ATCC (ATCC, Manassas, VA, USA). The cell line was isolated from the vascular endothelium of a White female patient. Cells were maintained in M199 with the supplementation of 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% amphotericin B, 2 mM glutamine (Biosera, Nuaille, France), and Endothelial Cell Growth Medium-2 (Lonza, Basel, Switzerland), at 37 °C, in a Galaxy 170R incubator under 5% CO2 (Eppendorf, Hamburg, Germany). Adhesion of the cells was support with a 0.1% gelatin solution.
9
Hypoxia-induced LSEC miR-9-5p regulation
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Briefly, LSECs (Ningbo Mingzhou Biotechnology Co. Ltd, MZ-M0395) were incubated in an Eppendorf Galaxy 170 R incubator under hypoxia conditions (5% CO2 and 95% N2 at 5 L/min) for 30 min till oxygen concentration reached 1%. The hypoxia mixture gas was continuously injected to maintain O2 concentration in the incubator below 1%. Then, cells were treated with glucose-free Dulbecco’s modified Eagle’s medium (DMEM) for 6 h, followed by HG (30 mmol/L glucose) DMEM medium containing 10% fetal bovine serum (FBS) at 5% CO2 for 12 h. The sequences of miR-9-5p mimic and miR-9-5p mimic NC were provided by the Shanghai GenePharma Co., Ltd (Shanghai, China). LSECs were inoculated into a 6-well plate 24 h before transfection. Upon reaching approximately 50% of cell confluence, the cells were incubated with 200 μL of transfection reagent and plasmids, as per the instructions in the manual from Lipofectamine 2000 transfection kit (11668-027; Invitrogen Inc., Carlsbad, CA, USA). The medium was changed after 6 h and collected after 48 h, according to the manufacturer’s instructions.
10
Shh-K562 Cell Line Generation
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K562 cell line was a kind gift from Dr. Sudhir Krishna’s lab, Bangalore, India. Shh-K562 cell line was made by transfecting Shh-Np containing pCDNA 3.1 plasmid using lipofectamine Reagent (ThermoFischer Scientific). The cells were cultured in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (Gibco, Life Technologies) with the addition of 1% Pen-strep (Gibco, Life Technologies) and maintained in a 5% CO2 humidified Galaxy 170 R incubator (Eppendorf) at 37 °C.
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