The most common and aggressive type of RCC exhibits loss of von Hippel-Lindau (VHL) factor and constitutive expression of hypoxic inducible factor (HIF); therefore, we chose 786-O (CRL-1932, ATCC, Germany) cells to establish RCC xenografts. Indeed, 786-O cells are characterized by a truncated form of HIF-1α and absence of VHL. In RCC, when an HIF-1α mutation is present, VEGF is controlled by constitutive expression of HIF-2α [79 (link)]. Cells were cultivated in high glucose RPMI-1640 medium (Sigma-Aldrich, Austria) supplemented with heat-inactivated fetal bovine serum (Gibco, Austria) and 100x penicillin/streptomycin amphotericin B solution (Lonza, Germany) diluted 1:100.
Penicillin streptomycin amphotericin b solution
Manufactured by Lonza
Sourced in United States, Austria
Penicillin/streptomycin amphotericin B solution is a liquid mixture of the antibiotics penicillin, streptomycin, and the antifungal agent amphotericin B. It is commonly used in cell culture applications to prevent bacterial and fungal contamination.
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Lab products found in correlation
Heparin, by STEMCELL (7 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Epidermal growth factor (egf), by Merck Group (6 mentions)
B27 neural supplement, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Ren cell vm human neural progenitor cells, by Merck Group (4 mentions)
Matrigel, by BD (4 mentions)
T25 cell culture flasks, by BD (3 mentions)
High glucose rpmi 1640 medium, by Merck Group (2 mentions)
Lps rs, by InvivoGen (2 mentions)
Dmem high glucose medium, by Merck Group (1 mentions)
Matrigel basement membrane matrix, by Corning (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mccoy s 5a medium, by Merck Group (1 mentions)
Sk n be, by Merck Group (1 mentions)
Glutamax, by Merck Group (1 mentions)
Ham s f12 medium, by Merck Group (1 mentions)
Eagle s minimum essential medium, by Merck Group (1 mentions)
Mem non essential amino acid solution, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
15 protocols using penicillin streptomycin amphotericin b solution
1
Establishment of RCC Xenograft Model
2
Breast Cancer Xenograft Generation
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MDA-MB-468 cell line (ATCC, HTB-132) was used for the generation of breast cancer xenografts. Cells were cultivated in high glucose DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with heat-inactivated fetal bovine serum (Gibco, Vienna, Austria) and penicillin/streptomycin amphotericin B solution (Lonza, Cologne, Germany).
3
Myosatellite Cell Culture Protocol
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The growth medium consisted of 20% FBS and 1% PSA antibiotics in Ham’s F-10 nutrient mix. The differentiation medium consisted of 2% FBS and 1% antibiotics (penicillin-streptomycin-amphotericin B solution, Lonza, Swiss) in Dulbecco’s Modified Eagle Medium (Gibco). Hanwoo myosatellite cells were cultured in hypoxic (2% O2, 5% CO2, 93% N2) and normoxic (20% O2, 5% CO2, 75% N2) conditions using growth medium (GM) for proliferation, and differentiation medium (DM) for differentiation. The atmospheric composition of the incubators (Hanwoo myosatellite cells were cultured in CelCuture® CO2 Incubators, ESCO, and Belgian Blue myosatellite cells were cultured in Great CO2 Incubator for Cultured Cells, Sanyo) were set by injecting CO2 and N2 gas. The satellite cells were seeded at 1800 cells/cm2 and incubated in a humidified incubator at 37 ℃. The satellite cells were cultured in GM until confluent on the 96-well plate or T25 flask and then differentiated into muscle myotubes in DM. Cells cultured with GM only were seeded on pre-coated bovine collagen type I flasks, and cells replaced with DM from GM were seeded into pre-coated Matrigel flasks (Cat # 354234, Matrigel® Basement Membrane Matrix, Corning®, Herndon, VA, USA).
4
Differentiation of ReN cell-derived NPCs
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ReN cell VM human neural progenitor cells (NPCs) were purchased from EMD Millipore (Billerica, MA, USA). The cells were plated onto BD Matrigel (BD Biosciences, San Jose, CA, USA)‐coated T25 cell‐culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in DMEM/F12 (Life Technologies, Grand Island, NY, USA) media supplemented with 2 mg heparin (StemCell Technologies, Vancouver, Canada), 2% (v/v) B27 neural supplement (Life Technologies, Grand Island, NY, USA), 20 mg EGF (Sigma–Aldrich, St Louis, MO, USA), 20 mg bFGF (Stemgent, Cambridge, MA, USA), and 1% (v/v) penicillin/streptomycin/amphotericin‐B solution (Lonza, Hopkinton, MA, USA) in a CO2 cell culture incubator. Cell‐culture media were changed every 3 d until cells were confluent. For 2D neuron/astrocyte differentiation, the cells were plated onto Matrigel‐coated microfluidic devices with DMEM/F12 differentiation media supplemented with 2 mg heparin, 2% (v/v) B27 neural supplement, and 1% (v/v) penicillin/streptomycin/amphotericin‐B solution without growth factors. Differentiation media was changed every 3–4 days.
5
Diesel Particulate Matter 2.5 Preparation
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Diesel particulate matter 2.5 (NIST SRM 1650b, National Institute of Standards and Technology, Gaithersburg, MD) stock solution (PM2.5 stock solution, 10 mg mL−1) was prepared in dimethyl sulfoxide (DMSO, Sigma‐Aldrich, St. Louis, MO) and sonicated for 30 min at room temperature (RT) to disperse the particles in the solution.[29 ] Afterward, PM2.5 solution was diluted in Dulbecco's modified Eagle medium (DMEM)/F‐12 (Thermo Fisher Scientific, Waltham, MA)‐based differentiation media consisting of: 2 µg mL−1 of heparin (STEMCELL Technologies, Vancouver, Canada); 2% v/v B27 neuronal supplement (Thermo Fisher Scientific); and 1% v/v penicillin–streptomycin–amphotericin‐B solution (Lonza, Basel, Switzerland). The final concentrations were varied depending on experimental settings. For control counterparts, DMEM/F‐12 differentiation medium was employed including 0.1% DMSO, 2 µg mL−1 of heparin, 2% v/v B27, and 1% v/v penicillin–streptomycin–amphotericin‐B solution without PM2.5 component.
6
Cell Culture Protocols for Various Cell Lines
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All media were supplemented with heat-inactivated fetal bovine serum (Gibco, Vienna, Austria) and penicillin/streptomycin amphotericin B solution (Lonza, Cologne, Germany).
All cell lines have been purchased from American Type Culture Collection. NB cell lines SH-SY5Y and SK-N-BE(2) were cultured in a 1:1 mixture of Ham’s F-12 medium (Sigma-Aldrich, Austria) and Eagle’s minimum essential medium (Sigma-Aldrich, Vienna, Austria). The mixture was supplemented with GlutaMAX (Gibco, Vienna, Austria) and MEM nonessential amino acid solution (Sigma-Aldrich).
RCC 786-O cells were cultured in a high glucose RPMI-1640 medium (Sigma-Aldrich), and RCC CAKI-2 cells were grown in McCoy’s 5a medium (Sigma-Aldrich).
The human embryonic kidney cell line HEK293 and the human normal dermal fibroblasts (HDFn) were grown in high glucose Dulbecco’s modified Eagle’s medium DMEM (Sigma-Aldrich), supplemented with GlutaMAX.
All cell lines have been purchased from American Type Culture Collection. NB cell lines SH-SY5Y and SK-N-BE(2) were cultured in a 1:1 mixture of Ham’s F-12 medium (Sigma-Aldrich, Austria) and Eagle’s minimum essential medium (Sigma-Aldrich, Vienna, Austria). The mixture was supplemented with GlutaMAX (Gibco, Vienna, Austria) and MEM nonessential amino acid solution (Sigma-Aldrich).
RCC 786-O cells were cultured in a high glucose RPMI-1640 medium (Sigma-Aldrich), and RCC CAKI-2 cells were grown in McCoy’s 5a medium (Sigma-Aldrich).
The human embryonic kidney cell line HEK293 and the human normal dermal fibroblasts (HDFn) were grown in high glucose Dulbecco’s modified Eagle’s medium DMEM (Sigma-Aldrich), supplemented with GlutaMAX.
7
Omicron BA.5.3.1.1.1 Spike-In Virus Protocol
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For the series of virus spike-in experiments (see below), we used SARS-CoV-2 cell culture isolate Slovakia/SK-BMC-BA43/2022 which was isolated from a COVID-19 patient from Slovakia in July 2022. The virus belongs to the BE.1.1 lineage (alias of BA.5.3.1.1.1; Omicron VOC) and is deposited in the European virus archive GLOBAL (available at https://www.european-virus-archive.com/virus/sars-cov-2-strain-slovakiask-bmc-ba432022-omicron-voc-be11-alias-ba53111 ). The virus stock was prepared in a Biosafety level 3 containment laboratory (Biomedical Research Center of the Slovak Academy of Sciences, Slovakia) by infecting Vero E6 cells (Vero C1008, ATCC CRL 1586) cultured in Eagle’s minimal essential medium (EMEM, Lonza, Switzerland) supplemented with 5% FBS (GIBCO, USA), Penicillin-Streptomycin-Amphotericin B Solution (10 ml/l, Lonza, Switzerland).
8
Neuron and Astrocyte Differentiation of ReN Cell-Derived NPCs
9
Neuron and Astrocyte Differentiation of ReN Cell-Derived NPCs
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ReN cell VM human neural progenitor cells (NPCs) were purchased from EMD Millipore (Billerica, MA, USA). The cells were plated onto BD Matrigel® (BD Biosciences, San Jose, CA, USA) coated T25 cell culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in DMEM/F12 (Life Technologies, Grand Island, NY, USA) media supplemented with 2 mg heparin (StemCell Technologies, Vancouver, Canada), 2% (v/v) B27 neural supplement (Life Technologies, Grand Island, NY, USA), 20 mg EGF (Sigma-Aldrich, St Louis, MO, USA), 20 mg bFGF (Stemgent, Cambridge, MA, USA) and 1% (v/v) penicillin/streptomycin/amphotericin-B solution (Lonza, Hopkinton, MA, USA) in a CO2 cell culture incubator. Cell culture media were changed every 3 days until cells were confluent. For 2D neuron/astrocyte differentiation, the cells were plated onto Matrigel®-coated microfluidic devices with DMEM/F12 differentiation media supplemented with 2 mg heparin, 2% (v/v) B27 neural supplement, and 1% (v/v) penicillin/streptomycin/amphotericin-B solution without growth factors. One half volume of the differentiation media was changed every 3–4 days for 3–9 weeks. LPS-RS was purchased from InvivoGen (San Diego, CA). LPS-RS was resuspended in dimethylsulphoxide (DMSO; 0.1 mg in 10 ml) followed by 1:10 dilution in differentiation culture medium. This stock solution of 22.2 mM was then diluted for cell treatment experiments.
10
Culturing Human Fetal Neural Progenitor Cells
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ReNcell CX immortalized cell line (SCC007, Millipore Sigma) derived from the cortical region of human fetal brain tissue was cultured to commit into neural progenitor cells (NPCs) according to the supplier’s protocol. The cells were plated onto BD Matrigel-coated T25 cell culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in DMEM/F12 (Life Technologies, Grand Island, NY, USA) media supplemented with 2 μg ml−1 heparin (StemCell Technologies, Vancouver, Canada), 2% (v/v) B27 neural supplement (Life Technologies, Grand Island, NY, USA), 20 μg ml−1 EGF (Sigma-Aldrich, St Louis, MO, USA), 20 μg ml−1 bFGF (Stemgent, Cambridge, MA, USA), and 1% (v/v) penicillin/streptomycin/amphotericin-b solution (Lonza, Hopkinton, MA, USA) in 5% CO2 incubator at 37 °C. Half of the cell culture medium was changed every 3 days until the cells were confluent.
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