Non-myocyte cells and cardiomyocytes were obtained by the Langendorff method using retrograde perfusion through the aorta. The heart was removed rapidly and retrograde-perfused under constant pressure (60 mmHg; 37 °C, 8 min) in Ca2+-free buffer (113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 5.5 mM glucose, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 12 mM NaHCO3, 10 mM KHCO3, 10 mM Hepes, 10 mM 2,3-butanedione monoxime, and 30 mM taurine). Digestion was initiated by adding a mixture of recombinant enzymes (0.2 mg/ml Liberase Blendzyme (Roche, 05401127001), 0.14 mg/ml trypsin (ThermoFisher, 15090046), and 12.5 μM CaCl2) to the perfusion solution. When the heart became swollen (10 min), it was removed and gently teased into small pieces with fine forceps in the same enzyme solution. Heart tissue was further dissociated mechanically using 2, 1.5, and 1 mm-diameter pipettes until all large heart tissue pieces were dispersed. The digestion buffer was neutralized with stopping buffer (10% fetal bovine serum (FBS; Capricorn, FBS-12A), 12.5 μM CaCl2). Cardiomyocytes were pelleted by gravity (7 times, 30 min each), and the supernatant was used as a source of non-myocyte cardiac cells [27 (link)].
Fbs 12a
Manufactured by Capricorn
Sourced in Germany
The FBS-12A is a laboratory equipment designed for specialized functions. It is a compact, versatile device intended for use in research and analytical settings. The core function of the FBS-12A is to perform specific tasks related to laboratory procedures. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Liberase blendzyme, by Roche (1 mentions)
Gentamycin, by Serva Electrophoresis (1 mentions)
Mem non essential amino acids solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Serva Electrophoresis (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
T4049, by Merck Group (1 mentions)
Geneticin, by Carl Roth (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Capricorn (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using fbs 12a
1
Isolation of Cardiac Cell Types
2
CaCo-2 and HEK293T cell cultivation
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CaCo-2 cells (kindly provided by Konstantin Sparrer, University Hospital Ulm, Germany) were cultivated in DMEM (Dulbecco’s Modified Eagle Medium DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS-12A; Capricorn Scientific, Ebsdorfergrund, Germany), 2 mM Gluta-MAX™ (35050061, Thermo Fisher Scientific), 25 mM HEPES (15630080, Thermo Fisher Scientific), 1× MEM Non-Essential Amino Acids Solution (11140050, Thermo Fisher Scientific), and 50 µg/mL gentamycin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany) and passaged every 2–3 days depending on confluence.
HEK293T T7/N cells (previously described in [19 (link)]) were cultivated in DMEM (Dulbecco’s Modified Eagle Medium DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS, 2 mM Gluta-MAX™, 25 mM HEPES, 5 µg/mL blasticidin (asnt-bl-1, InvivoGen, San Diego, CA, USA), and 2 µg/mL puromycin (SC-1080713, Santa Cruz Biotechnology, Dallas, TX, USA).
All cells were incubated at 37 °C, 5% CO2, and 80% relative humidity.
HEK293T T7/N cells (previously described in [19 (link)]) were cultivated in DMEM (Dulbecco’s Modified Eagle Medium DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS, 2 mM Gluta-MAX™, 25 mM HEPES, 5 µg/mL blasticidin (asnt-bl-1, InvivoGen, San Diego, CA, USA), and 2 µg/mL puromycin (SC-1080713, Santa Cruz Biotechnology, Dallas, TX, USA).
All cells were incubated at 37 °C, 5% CO2, and 80% relative humidity.
3
Engineered HeLa Cell Lines for CERT Study
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The HeLa-mCAT #8 clone, a derivative of the HeLa ATCC CCL-2, which stably expresses the mouse ecotropic retroviral receptor mCAT1 (Yamaji et al., 2010 (link)), was used as the parental cell line in this study. The HeLa CERT KO #14 clone was established as described previously (Yamaji and Hanada, 2014 (link)). The HeLa CERT KO/HA-CERT (Goto et al., 2022a (link)) was used as a cell line ectopically expressing HA-tagged CERT in CERT KO cells.
DMEM High Glucose (044-29765; Fujifilm Wako Pure Chemicals Corporation) supplemented with 10% FBS (172012; Sigma-Aldrich, or FBS-12A, Capricorn Scientific) and penicillin–streptomycin (P4333; Sigma-Aldrich) was used for cell culture, unless otherwise mentioned. HeLa cells were maintained in the culture medium in a 5% CO2 atmosphere, 100% humidity at 37°C.
DMEM High Glucose (044-29765; Fujifilm Wako Pure Chemicals Corporation) supplemented with 10% FBS (172012; Sigma-Aldrich, or FBS-12A, Capricorn Scientific) and penicillin–streptomycin (P4333; Sigma-Aldrich) was used for cell culture, unless otherwise mentioned. HeLa cells were maintained in the culture medium in a 5% CO2 atmosphere, 100% humidity at 37°C.
4
Propagation of HCMV in Human Foreskin Fibroblasts
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Primary human foreskin fibroblasts (HFFs, own cell cultures repository of M.M. laboratory) and HFF-UL50 cells [33 (link)] were maintained at 37 °C, 5% CO2, and 80% humidity in MEM supplemented with 10% fetal bovine serum (FBS, FBS-12A, Capricorn Scientific, Ebsdorfergrund, Germany), 1×GlutaMAX™ (35050038, Thermo Fisher Scientific, Waltham, MA, USA), and 10 µg/ml of gentamicin (22185.03, SERVA, Heidelberg, Germany). For the cultivation of HFF-UL50, tetracycline negative FBS (FBS-TET-12A, Capricorn Scientific) was used and, additionally, 500 µg/ml of geneticin was added (G418, 10131035, Thermo Fisher Scientific). The expression of pUL50 in the HFF-UL50 cells was induced by addition of 500 ng/ml of doxycycline (D9891, Sigma-Aldrich, St. Louis, MO, USA) which was refreshed at least every 3rd day (d). For HCMV infection of HFF-UL50, the cells were induced with dox one d prior to infection to induce pUL50 expression. HFF or HFF-UL50 cells were inoculated with stocks of HCMV AD169 (WT) or AD169-derived recombinant ΔUL50 viruses [33 (link)] with equal genome amounts. After incubation for 90 min at 37 °C, the inoculum virus was replaced by fresh medium. Cells or supernatants were harvested or fixed at indicated time points for further analyses.
5
Cell Culture of Human Breast Cancer and HEK293T Lines
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All the human BC (MDA-MB-231, MCF7, BT-474, Hs578T, CAL-51) and the HEK293T cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (41966 Gibco, Waltham, MA, USA), supplemented with 10% foetal bovine serum (FBS-12A Capricorn Scientific, Birmingham, England), 100 U/mL penicillin, and 100 µg/mL streptomycin (15140122 Thermo Fisher, Waltham, MA, USA), and maintained at 37 °C in 5% CO2 humidified air. MDA-MB-231 cells were authenticated on the basis of viability, recovery, growth, and morphology by ATCC on 24 September 2018, and all cell lines were tested for mycoplasma contamination using the EZ-PCR™ Mycoplasma Detection Kit (20-700-20BI).
6
SARS-CoV-2 Antiviral Activity Evaluation
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Vero E6 cells (CRL-1586, ATCC maintained at 37 °C; 5% CO2) were seeded at 2.104 cells per well in a 96-well plate in Dulbecco’s Modified Eagle’s Medium, 5% fetal bovine serum (FBS-12A, Capricorn Scientific, Clinisciences). For cyclophosphamide antiviral activity evaluation, cells were treated with 0.15 mg/mL (corresponding to the maximum dose potentially present in hamsters) or 0.45 mg/mL cyclophosphamide diluted in sterile PBS. For IcatCXPZ-01 antiviral activity evaluation, cells were treated with 4.5 µg/mL (corresponding to the maximum dose potentially present in hamsters) or 13.5 µg/mL IcatCXPZ-01 diluted in 10% dextrin, citrate buffer 50 mM, pH = 5. Cells were treated with PBS as control (n = 6 for each condition) and molecule cytotoxicity was tested as well without infection at the highest used concentration. All treatments were started one hour prior to infection with SARS-CoV-2 strain France/IDF0372/2020 at 5 × 103 pfu per well diluted in DMEM, 10% fetal bovine serum. Loss of cell viability reflecting the efficiency of viral infection was measured 3 days after infection by adding 100 µL Cell Titer-Glo reagent to each well (CellTiterGlo Luminescent Cell Viability Assay, Promega #G7571), according to the manufacturer’s protocol. Cell luminescence of each well was then quantified using an Infinite M200Pro TECAN and normalized to the control condition.
7
HeLa Cell Cultivation Protocol
8
Isolation and Differentiation of Murine Bone Marrow-Derived Macrophages
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Femur and tibia of PdgfbWT/WT and Pdgfbret/ret mice on standard laboratory diet were dissected. Bone marrow cells were isolated by flushing bones with PBS. Single cell suspensions were obtained by passing cells through a 70 µm cell strainer.
Bone marrow cells were cultured on non-tissue-culture-treated petri dishes in RPMI 1640 medium (72400047, Gibco, Waltham, MA, USA) or DMEM medium (31966021, Gibco) with 15% cell line L929-conditioned medium (LCM), 10% heat-inactivated fetal calf serum (FCS, FBS-12A, Capricorn Scientific, Ebsdorfergrund, Germany) and 1% Penicillin Streptomycin (P/S, 15070-063, Gibco). LCM was added to ensure differentiation of bone marrow-derived monocytes to macrophages (BMDMs). After 7 day differentiation, BMDMs were detached with lidocaine and generally plated onto non-tissue culture treated plates for various assays. Prior to the addition of stimuli, BMDMs were always allowed to attach overnight.
Bone marrow cells were cultured on non-tissue-culture-treated petri dishes in RPMI 1640 medium (72400047, Gibco, Waltham, MA, USA) or DMEM medium (31966021, Gibco) with 15% cell line L929-conditioned medium (LCM), 10% heat-inactivated fetal calf serum (FCS, FBS-12A, Capricorn Scientific, Ebsdorfergrund, Germany) and 1% Penicillin Streptomycin (P/S, 15070-063, Gibco). LCM was added to ensure differentiation of bone marrow-derived monocytes to macrophages (BMDMs). After 7 day differentiation, BMDMs were detached with lidocaine and generally plated onto non-tissue culture treated plates for various assays. Prior to the addition of stimuli, BMDMs were always allowed to attach overnight.
9
Wild Boar Tissue Sampling and DNA Extraction
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Tissue samples from 63 shot free-living wild boars were collected from August to November 2018. The animals originated from the South Banat District, i.e. municipalities of Pančevo, Bela Crkva, Plandište, Vršac, and Alibunar. The samples were collected in agreement with the local hunting organizations. In total, this examination involved 122 tissue samples: 61 samples of spleen, 43 tonsil samples, and 18 lymph node samples. The specimens were immersed into 2 ml of minimum essential medium (MEM, Capricorn Scientifi c, Germany) with 2% fetal calf serum (FBS-12A, Capricorn Scientifi c, Germany) supplemented with antibiotics and transported to the laboratory on ice. All organ samples were homogenized in phosphate buffered saline (PBS 7.2) and centrifuged for 10 min at 5000 RPM. Deoxyribonucleic acid (DNA) was extracted from the cell debris using GeneJET Genomic DNA Purifi cation Kit (Thermo Scientifi c, USA) according to the manufacturer's instructions. The extracted DNA was stored at -20°C pending testing.
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