Following cell expansion, 1 × 105 CD29+ cells were seeded in a 96-well U-bottom plate (IWAKI, Chiyoda City, Japan) to form spheroids. Subsequently, the plate was centrifuged at 4 × 400× g for 3 min. After 3 days of incubation, spheroids were cultured in a myogenic differentiation medium comprising DMEM with GlutaMax (Life Technologies) supplemented with 5% horse serum (Life Technologies) for 5 days. Next, spheroids were cultured in adipogenic differentiation medium comprising DMEM with GlutaMax (Life Technologies), supplemented with 5% horse serum (Life Technologies), 1 μM IBMX, 1 μM dexamethasone, 1 mM insulin-transferrin-selenium, 50 μM indomethacin, and 500 μM oleic acid (Tokyo Chemical Industry) for 3 days. Subsequently, the medium was replaced with DMEM supplemented with GlutaMax (Life Technologies), 5% horse serum (Life Technologies), 1 μM IBMX, 1 μM dexamethasone, 50 μM indomethacin, and 500 μM oleic acid. Cells were cultured for 2 weeks to facilitate adipogenic differentiation. All chemicals used in the experiment were for research purposes only.
Horse serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, New Zealand, Japan, China, France, Australia, Italy, Spain, Switzerland, Canada, Netherlands, Denmark, Austria, Belgium, Ireland, Israel, Brazil
Horse serum is a biological fluid derived from the blood of horses. It contains a complex mixture of proteins, including immunoglobulins, hormones, and other biomolecules. Horse serum is commonly used as a supplement in cell culture media to support the growth and maintenance of various cell types.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (760 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (472 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (398 mentions)
Hydrocortisone, by Merck Group (320 mentions)
Cholera toxin, by Merck Group (301 mentions)
Dmem f12, by Thermo Fisher Scientific (254 mentions)
Insulin, by Merck Group (253 mentions)
Penicillin, by Thermo Fisher Scientific (221 mentions)
Streptomycin, by Thermo Fisher Scientific (213 mentions)
L glutamine, by Thermo Fisher Scientific (155 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (143 mentions)
Glutamax, by Thermo Fisher Scientific (98 mentions)
Mcf 10a, by American Type Culture Collection (94 mentions)
Epidermal growth factor (egf), by Merck Group (92 mentions)
Mcf 10a, by Thermo Fisher Scientific (79 mentions)
Insulin, by Thermo Fisher Scientific (75 mentions)
Penicillin streptomycin, by Merck Group (73 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (72 mentions)
Rpmi 1640, by Thermo Fisher Scientific (67 mentions)
Mda mb 231, by American Type Culture Collection (61 mentions)
2 251 protocols using horse serum
1
Adipogenic Differentiation of CD29+ Cells
2
Rab14 Colocalization with Lysosomal Membrane
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At the indicated time points post infection, coverslips were washed with PBS and permeabilized with 0.1% (w/v) saponin (Sigma) in PBS for 30 min. Coverslips were then incubated for 120 min with anti-Rab14 (4 μg/mL in 0.1% (v/v) horse serum (Gibco), 0.1% (w/v) saponin in PBS; clone D-5, murine IgG1, sc-271401, Santa Cruz), washed with PBS, followed by a 45 min incubation with anti-mouse IgG H&L labelled with AlexaFluor 647 (10 μg/mL in 0.1% (v/v) horse serum (Gibco), 0.1% (w/v) saponin in PBS, polyclonal, donkey IgG, ab150111, Abcam). Coverslips were washed with PBS, and then incubated with anti-Lamp1 (1 μg/mL in 0.1% (v/v) horse serum (Gibco), 0.1% (w/v) saponin in PBS, clone 1D4B, rat IgG2a, sc-19992, Santa Cruz) for 20 min, washed with PBS, and incubated for 20 min with anti-rat IgG H&L labelled with AlexaFluor 568 (10 μg/mL in 0.1% (v/v) horse serum (Gibco), 0.1% (w/v) saponin in PBS, polyclonal, goat IgG, A11077, Life Technologies). Coverslips were mounted in microscope slides with ProLong Gold antifade mountant (Invitrogen), and visualised on a TCS-SP5 inverted microscope (Leica Biosystems). To determine the percentage of the Lamp1 positive KCV that co-localized with Rab14, KCVs of at least 100 infected cells from three independent experiments were analysed.
3
Isolation and Culture of Sheep Skeletal Muscle Stem Cells
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Human HEK293 and murine C2C12 myoblast cells were purchased from the China Infrastructure of Cell Line Resource. C2C12 myoblasts were cultured in growth medium (GM) consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% FBS (Gibco, USA) at 37 °C with 5% CO2. Myogenic differentiation was induced by a differentiation medium (DM) with DMEM supplemented with 2% horse serum (Gibco, USA). HEK293 cells were maintained in DMEM with 10% FBS at 37 °C with 5% CO2.
The sheep skeletal muscle SCs were enzymatically isolated from leg muscle tissues obtained from five Duolang sheep fetuses at 90 days, using a two-step digestion method, as described previously36 (link). Briefly, leg muscle tissues were excised, cut into small pieces, digested with 0.1% type I collagenase (Sigma-Aldrich, USA) for 1 h, and then digested with 0.25% trypsin (Gibco, USA) for 20 min. The samples were vortexed every 10 min and filtered through a 200-mesh sieve, and SCs were purified using the differential adhesion method49 (link). Sheep skeletal muscle SCs were proliferated in GM consisting of DMEM/F-12 (Gibco, USA) supplemented with 20% FBS and 10% horse serum in a collagen type 1-coated plate, and then differentiated using DMEM/F-12 with 2% horse serum.
The sheep skeletal muscle SCs were enzymatically isolated from leg muscle tissues obtained from five Duolang sheep fetuses at 90 days, using a two-step digestion method, as described previously36 (link). Briefly, leg muscle tissues were excised, cut into small pieces, digested with 0.1% type I collagenase (Sigma-Aldrich, USA) for 1 h, and then digested with 0.25% trypsin (Gibco, USA) for 20 min. The samples were vortexed every 10 min and filtered through a 200-mesh sieve, and SCs were purified using the differential adhesion method49 (link). Sheep skeletal muscle SCs were proliferated in GM consisting of DMEM/F-12 (Gibco, USA) supplemented with 20% FBS and 10% horse serum in a collagen type 1-coated plate, and then differentiated using DMEM/F-12 with 2% horse serum.
4
Isolating Human Skeletal Muscle Cells
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Human skeletal muscle tissue was first minced and incubated in collagenase II (750 U/ml, Worthington Biochemical) in Ham’s F10 medium supplemented with 10% horse serum (Invitrogen) and 1% penicillin-streptomycin (Omega Scientific) at 37°C for 90 min in a shaking water bath (60 rpm). The digested muscle was washed in F10 with 10% horse serum and digested further in a shaking water bath at 37°C for 30 min in collagenase II (100 U/ml) and dispase (2 U/ml, Gibco) in F10 with 10% horse serum. Subsequent steps in the isolation procedure are detailed in the Supplemental Experimental Procedures .
5
Immunofluorescence Staining for Viral Infection
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Cells were fixed at the end of respective incubation time in 3% PFA solution for at least 10 min at room temperature before permeabilization with 0.2% Triton X-100 in PBS and blocking by 5% horse serum (ThermoFisher Scientific, 26050-088) in PBS at room temperature for at least 1 h. For co-infection and sequential infection experiments, HEV capsid was stained with rabbit anti-HEV-ORF2 serum (kind gift of Prof. Rainer G. Ulrich, Friedrich Loeffler Institute, Germany, 1/4000 in PBS supplemented with 5% horse serum), while for super-infection experiments, HEV was stained with rabbit anti-GFP (Invitrogen, A11122, 1/1000 in PBS supplemented with 5% horse serum). HCV was stained with murine anti-HCV-NS5A-9E10 antibody (kind gift of Prof. Charles M. Rice, Rockefeller University, New York, USA, 1/10,000 in PBS supplemented with 5% horse serum), all overnight at 4 °C. HEV secondary staining was performed with Alexa 488-labeled goat anti-rabbit IgG (Invitrogen A11008, 1/1000 in PBS supplemented with 5% horse serum), HCV with Alexa 555-labeled donkey anti-mouse IgG (Invitrogen, A32773, 1/1000 in PBS supplemented with 5% horse serum). Plates were imaged at an Olympus IX81 microscope (Olympus) (transfection experiments) or a Keyence BZ X810 (Keyence Deutschland GmbH, Neu-Isenburg, Germany).
6
Multilineage Stem Cell Differentiation
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Cells were stimulated with specific differentiation media Stem Pro differentiation kits (Life technologies, USA) following manufactures protocol. For myogenic differentiation two different media were prepared, cells were grown in DMEM media with 2% horse serum (Life technologies, USA) and 1% l-glutamin (Life technologies, USA) for 3 days and then changed to DMEM with 2% horse serum, 1%l-glutamin 1ng/ml bFGF (Peprotech, USA) and 0.4μg/ml dexamethasone (Sigma Aldrich, USA). Adipogenic differentiation was assessed with Red oil O (Sigma Aldrich, USA) staining for fat vacuoles; Myogenic differentiation was assessed using staining for α-SMA; Osteogenic differentiation was demonstrated by assessing alkaline phosphotase activity (BCIP/NBT, Thermo Scientific, USA); Chondrogenic differentiation was demonstrated via staining for Toluidine Blue (Sigma Aldrich, USA).
7
Primary Cultured Cortical Neurons from Mice
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Primary cultured cerebral cortical neuronal cells were prepared from ddY mice (Japan SLC, Hamamatsu, Japan) at embryonic day 14 (E14) as described previously (Tohda et al., 2012 (link)). Eight-well chamber slides coated with 5 µg/ml poly-D-lysine (PDL; Sigma-Aldrich, St. Louis, MD, USA) were used for cultures. The cells were cultured on PDL-coated slides with neurobasal medium (Life Technologies, Carlsbad, CA, USA) containing 12% horse serum (Life Technologies), 0.6% glucose, 2 mM L-glutamine (HS medium) at 37° in a humidified incubator with 10% CO2. Four hours later, the medium was replaced with fresh neurobasal medium containing 2% B-27 supplement (Life Technologies) without horse serum (B27 medium).
8
Isolation and Differentiation of Murine Satellite Cells
9
Establishment and Maintenance of Breast Cell Lines
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MCF10A cells were obtained from the American Type Culture Collection (ATCC) and were maintained in DMEM/F12 (Lonza, Allendale, NJ) supplemented with 5 % horse serum (Life Technologies, Carlsbad, CA), 20 ng/ml EGF (Life Technologies), 0.5 μg/ml hydrocortisone (Sigma, St. Louis, MO), 100 ng/ml cholera toxin (Sigma), 10 μg/ml insulin (Akron Biotech, Boca Raton, FL), and 1 % penicillin-streptomycin (Life Technologies). MCF10AT, MCF10DCIS and SUM225 cells were obtained from Dr. Fariba Behbod (University of Kansas Medical Center). MCF10AT cells were maintained in the same media as used for the MCF10A cells. MCF10DCIS cells were maintained in DMEM/F12, 5 % horse serum, and antibiotic/antimycotic (Life Technologies). 293 T cells were obtained from ATCC and maintained in D-MEM supplemented with 10 % FBS and 1 % penicillin-streptomycin (Life Technologies). MCF7 and MDA-MB-231 cells were obtained from ATCC and maintained as recommended. For treatment of cells with EREG, cells were treated with 10 ng/ml recombinant human EREG (rhEREG) (1195-EP-025/CF; R&D Systems, Minneapolis, MN). For inhibitor studies, cells were treated with the indicated amounts of dovitinib (TKI-258, LC Laboratories) or the appropriate amounts of solvent control.
10
Quantifying Viral Protein Localization and Infectivity
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At 48 hours p.t. or 7 days postinfection, transfected cells previously seeded onto coverslips or on 96‐well‐plates were fixed with 3% PFA in PBS and permeabilized with 0.5% Triton X‐100 in PBS. After blocking of nonspecific binding with 5% goat serum (Sigma‐Aldrich) and horse serum (Gibco) in PBS, immunostaining of ORF2‐encoded capsid protein was performed. A 1:1,000 PBS‐diluted mouse antibody directed against the ORF2‐encoded protein (amino acids 434‐457, clone 1E6; LifeSpan BioSciences, Inc., Seattle, WA) or a 1:500 dilution of an ORF2‐specific rabbit hyperimmune serum (kindly provided by R. Ulrich, Friedrich Loeffler Institute, Greifswald, Germany) was added. A fluorescently labelled goat antibody (AlexaFluor 488; Life Technologies) was used at a dilution of 1:1,000 in PBS with 5% goat or horse serum to detect bound primary antibodies. DNA was labelled with 4′,6′‐diamidino‐2‐phenylindole (Life Technologies). For quantification of virus infectivity, wells were manually observed for specific fluorescence, and the presence of fluorescent foci was recorded. A fluorescent focus was defined as a minimum of two adjacent cells showing clear intracytoplasmic fluorescence. The number of fluorescent foci was counted in the highest dilution showing fluorescence, and the respective focus‐forming units (FFU) were calculated. The limit of detection was 1 FFU.
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