Cell lines and organotypic explant slices were maintained in standard tissue culture flasks or dishes. Primary hepatocytes were plated into collagen I–coated 175-cm2 flasks (Corning, #354487). Cells (except U-2 OS NR1D1::VNP cells) and organotypic slices were maintained in complete culture medium: Dulbecco’s modified Eagle’s medium (DMEM) [glucose (4500 mg/liter) and l -glutamine; Thermo Fisher Scientific #41965039], supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml)/streptomycin (100 μg/ml) (P/S), and 25 mM Hepes buffer (in-house). Fluorescent reporter cells were maintained in RPMI 1640 (l -glutamine; Capricorn, #RPMI-A) with 10% FBS and 1% P/S. Organoids were maintained in specialized media formulations as indicated in the Supplementary Materials. Spheroids were embedded in Cultrex growth factor–reduced basement membrane matrix (Trevigen, #3433-001-01) and maintained in complete culture medium. All model systems were maintained under standard tissue culture conditions in humidified 5% CO2 at 37°C tissue culture incubators.
175 cm2 flask
Manufactured by Corning
Sourced in United States
The 175 cm2 flasks are a standard laboratory equipment used for cell culture and tissue engineering applications. These flasks provide a consistent and reliable growth surface for a variety of cell lines. The 175 cm2 surface area offers ample space for cell proliferation and expansion.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Microfuge 22r centrifuge, by Beckman Coulter (3 mentions)
24 well culture dishes, by Greiner (1 mentions)
Dmem f12, by Corning (1 mentions)
Falcon tube, by Corning (1 mentions)
Nystatin, by Merck Group (1 mentions)
Mem e, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Fresh bone marrow aspirate, by Lonza (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Murine gm csf, by R&D Systems (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
12 protocols using 175 cm2 flask
1
Standardized Culture Conditions for Cell Models
2
Quantification of Bacterial Adherence and Invasion in Human Bronchial Epithelial Cells
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Human bronchial epithelial 16HBE14 cells (Gruenert et al., 1988 (link)), kindly provided by Dr. Kirsten Spann (Queensland University of Technology), were seeded in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (sMEM) at an approximate density of 2 ∗ 105 cells/ml into 24-well culture dishes (Greiner Bio-One, Kremsmünster, Austria) (1 ml/well) for adherence and invasion assays or into 175-cm2 flasks (Corning, Tewksbury, MA, United States) (40 ml/flask) for maintenance. Confluent 16HBE14 monolayers were washed once with prewarmed sMEM and then infected with Hi2019WT, Hi2019ΔdmsA, or Hi2019ΔdmsA_c diluted in sMEM to 2 ∗ 107 bacteria/ml, giving a multiplicity of infection (MOI) of 1:100 (epithelial cells: bacteria). Bacterial adherence and invasion were determined as described previously (Dhouib et al., 2015 (link), 2016 (link)). Determination of intracellular bacteria used a standard gentamicin-protection assay (St Geme and Falkow, 1990 (link)). In brief, infected cells were washed to removed planktonic and loosely adherent bacteria and incubated for 1 h in sMEM containing 50 μg/ml gentamicin followed by saponin lysis and serial dilution for determination of bacterial CFU present. Assays used at least three biological replicates; replicate experiments were carried out on different days.
3
Serum-free Conditioning of SW480 Cells
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A total of 2 × 107 SW480 cells were cultured in DMEM/F-12 supplemented with 10% of FBS in 175 cm2 flasks (Corning, Inc.). When cells reached 80% confluence, the medium was aspirated, and the flasks were washed with sterile PBS to remove serum components. Next, 20 ml fresh serum-free DMEM/F12 was added and further cultured for 48 h at 37 °C in the presence of 5% CO2. After 48 h, the conditioned medium was collected in 50 ml Falcon tubes (Corning Life Sciences) using a sterile pipette.
4
Propagation of Pseudorabies Virus NIA3 in PK-15 Cells
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Pseudorabies virus strain NIA3 [32 (link)], kindly supplied by Joan Plana (Fort Dodge Veterinaria, Vall de Bianya, Spain), was propagated in the PK-15 cell line (provided by the Institute of Virology (UE and OIE Reference Laboratory for CSFV, Hannover), using a standard growth media (SGM) containing minimum essential medium eagle (MEM-E; ThermoFisher, Waltham, MA, USA) supplemented with 1% penicillin 10,000 U/mL and streptomycin 10 mg/mL (ThermoFisher), 0.5% Nystatin 10,000 IU/mL (Sigma-Aldrich), 1% L-glutamine 200 mM (ThermoFisher) and 5% of heat inactivated fetal bovine serum tested free for virus and antibodies against pestiviruses (FBS; BioWest, Miami, FL, USA). PK-15 cells were grown in 175-cm2 flasks (Corning, Corning, NY, USA), and when cells were confluent, the medium was discarded and adsorption was performed at a MOI 0.01. Virus stock was produced in the same cells to obtain 84 mL with a viral titer of 108.95 TCID50/mL that was used to inoculate 24 L of bovine plasma, achieving a final viral titer of 106.5 TCID50/mL.
5
Isolation and Expansion of hMSCs
6
Cell Line Cultivation and Verification
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BeWo (ATCC CCL-98), a human placental trophoblast cell line (Pattillo and Gey 1968 (link)) and K-562 (ATCC CCL-243), a hematopoietic stem cell line (Lozzio and Lozzio 1975 (link)), were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells used in experiments were within twenty passage numbers from arrival into the laboratory and were routinely verified by their short tandem repeat profiles using fragment analysis (ABI 3730XL DNA Analyzer, Applied Biosystems, Waltham, MA) at the University of Michigan Advanced Genomics Core. Unless otherwise noted, all media were supplemented with 10% (v/v) HI-FBS and 1% (v/v) of 10,000 U/mL P/S. IMDM and F12-K Nutrient Mixture Kaighn’s Modification with (+) L Glutamine were used as the media for culturing for K-562 cells and BeWo cells, respectively. All cells were plated at a 100,000 cells/mL in 25 mL of media in 175 cm2 flasks (Corning Inc., Corning, NY) and subcultured at 70–80% confluence. Unlike BeWo cells, K-562 cells are suspension cells and do not require 0.25% trypsin-EDTA for detachment in subculture. Cell cultures were maintained in a 5% CO2, 37°C controlled and humidified incubator. This work with human cell cultures was approved by the University of Michigan Institutional Biosafety Committee (IBCA00000100).
7
Pig Intestinal Epithelial Cell Culture
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All chemicals were purchased from Sigma–Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise stated. All cell culture experiments were conducted using pig intestinal epithelial cells (DSMZ, Braunschweig, Germany) in the cell culture facility located in Kemin Industries (Des Moines, IA). Cells were cultured in Dulbecco’s modified Eagle medium-low glucose supplemented with 10% fetal bovine serum, 1% antibiotics (Penicillin, Streptomycin, and Amphotericin B), and 5 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ) in a humidified atmosphere with 5% CO2 at 37.0 °C. Passages 10 to 15 of the cells were used for the experiment. Cells were maintained in 175 cm2 flasks (Corning Inc., Corning, NY) with the media being changed every third day until they were plated in Transwell plates.
8
Isolation and Expansion of Human Mesenchymal Stem Cells
9
Isolation of Murine Glial Cells
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Primary cultures of mixed murine glial cells were prepared as previously described using a modified protocol.38 ,48 (link) Briefly, brains from C57/BL6 P2 neonatal pups (purchased from Charles River, Saffron Walden, United Kingdom) were extracted, rolled across sterile filter paper to remove vasculature and meninges, and were mechanically dissociated. Cells were resuspended in 40 mL per 175 cm2 flask (Corning, Amsterdam, Netherlands) and were maintained in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen, Sigma Aldrich, Gillingham, United Kingdom) and incubated at 37°C (5% CO2/95% O2). A week later, 5 ng/mL of murine GM-CSF (R&D systems, Abingdon, United Kingdom) in fresh media was added to the flasks to expand yields, and the cells were maintained for a further week.48 (link)Microglia were harvested by overnight shaking in an orbital shaker incubator (37°C, 180 rev/min), and the supernatant was collected from the flasks to obtain the dissociated microglia. Trypsin was subsequently added to lift the astrocytic layer. Microglia and astrocytes cells were plated as required for further experiments.
10
Isolation and Assay of Cell Organelles
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The isolation
and assays were
performed similarly to that previously described.62 (link),63 (link) In brief, cells were grown to confluency with complete medium in
a 175 cm2 flask (Corning) before growth media was removed
and replaced with serum-free DMEM for 2 h. To neutralize endosomes
prior to lysis, FCCP was added to the cellular media to reach a final
concentration of 1 μM. Cells were incubated with FCCP for 15
min before cells were scrapped from the plate and pelleted at 1000g for 5 min. The media was discarded and cells were resuspended
in HEK assay buffer (20 mM HEPES, 5 mM glucose, 50 mM sucrose, 50
mM KCl, 90 mM potassium gluconate, 1 mM EGTA, Pierce protease inhibitor
mini tablet, pH = 7.4) and were then lysed by passage through a 22
gauge needle 10–15 times. Lysates were then centrifuged at
10,000g for 30 s with a Beckman Coulter Microfuge
22R centrifuge. The supernatant was removed and centrifuged at 14,500g for 20 min. The remaining supernatant was discarded, and
the pellet was resuspended in HEK assay buffer.
and assays were
performed similarly to that previously described.62 (link),63 (link) In brief, cells were grown to confluency with complete medium in
a 175 cm2 flask (Corning) before growth media was removed
and replaced with serum-free DMEM for 2 h. To neutralize endosomes
prior to lysis, FCCP was added to the cellular media to reach a final
concentration of 1 μM. Cells were incubated with FCCP for 15
min before cells were scrapped from the plate and pelleted at 1000g for 5 min. The media was discarded and cells were resuspended
in HEK assay buffer (20 mM HEPES, 5 mM glucose, 50 mM sucrose, 50
mM KCl, 90 mM potassium gluconate, 1 mM EGTA, Pierce protease inhibitor
mini tablet, pH = 7.4) and were then lysed by passage through a 22
gauge needle 10–15 times. Lysates were then centrifuged at
10,000g for 30 s with a Beckman Coulter Microfuge
22R centrifuge. The supernatant was removed and centrifuged at 14,500g for 20 min. The remaining supernatant was discarded, and
the pellet was resuspended in HEK assay buffer.
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