Demyelinating rat cerebellar slice cultures were prepared as previously described (Birgbauer, Rao, & Webb, 2004 ). Briefly, P9 rat cerebellum was isolated and 300 μm sections were obtained using McIlwain tissue chopper (McIlwain, San Antonio, TX). Slices were then incubated at 37°C with 5% CO2 in organotypic slice medium (50% BME [Gibco], 25% heat‐inactivated horse serum [Gibco], 25% HBSS [BME, Gibco], 5 mg/mL glucose, 1× glutamax [Gibco], and 1× Mycozap‐PR plus [Lonza]). After a week in culture, slices were demyelinated with 0.5 mg/mL lysolecithin for 16 hr and then incubated in lysolecithin‐free medium to allow remyelination.
Heat inactivated horse serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
Heat-inactivated horse serum is a cell culture supplement derived from the blood of horses. It has been subjected to a heat treatment process to inactivate any potential pathogens. This product is designed to support the growth and maintenance of cell lines in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (31 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (28 mentions)
Penicillin, by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
Glutamax, by Thermo Fisher Scientific (15 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (14 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (13 mentions)
Trypsin, by Thermo Fisher Scientific (12 mentions)
Neurobasal medium, by Thermo Fisher Scientific (10 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Insulin, by Merck Group (6 mentions)
B27 supplement, by Thermo Fisher Scientific (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (5 mentions)
Glutamine, by Thermo Fisher Scientific (5 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Poly d lysine, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
125 protocols using heat inactivated horse serum
1
Demyelinating Cerebellar Slice Cultures
2
Culturing Diverse Hematological Cell Lines
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DC4 + human primary AML samples and normal peripheral blood mononuclear cells (PBMCs) were obtained from residual samples using a protocol approved by the Institutional Review Board of Stony Brook University. THP-1, U937, TALL104, and NK-92 cell lines were obtained from ATCC (Manassas, VA, USA). MOLM-13 was obtained from AddexBio (San Diego, CA, USA) T cells were cultured in filtered T cell media, defined as 50% AIM V, 40% RPMI 1640 and 10%FBS, with 1% Pen/Strep (all Gibco, Waltham, MA, USA) and supplemented with IL-2 (300 IU/mL; Peprotech, Rocky Hill, NJ, USA), unless otherwise specified. NK-92 cells were cultured in filtered NK cell media, defined as alpha-MEM without ribonucleosides and deoxyribonucleosides with 2mM L-glutamine, 1.5 g/L sodium bicarbonate (Gibco), 12.5% heat-inactivated horse serum (Gibco), 12.5% heat-inactivated FBS (Atlanta Biologicals, Atlanta GA, USA), 1% Pen/Strep (Gibco), 0.2% inositol (Sigma), 0.02% folic acid (Fisher), and 50 uM beta-mercaptoethanol (Fisher), supplemented with IL-2 (300 IU/mL), unless otherwise specified. THP-1, U937, and MOLM-13 cell lines were cultured in RPMI, 10% FBS, 1% Pen/Strep (Gibco). TALL104 cells were cultured in IMDM adding 300 IU/ml recombinant human IL-2, 2.5 mg/ml human albumin, 0.5 mg/ml D-mannitol, and 20% FBS.
3
Differentiation of C2C12 Myoblasts
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Mouse skeletal muscle cell lines (C2C12 myoblasts) were obtained from Shanghai FuHeng Biology (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Co., Ltd., Grand Island, NY, United States) containing 10% (v/v) fetal bovine serum (Gibco) and 1% (w/v) penicillin–streptomycin (P/S; NCM Biotech, Suzhou, China) in a humidified incubator under 5% (v/v) CO2 at 37°C. To initiate differentiation, cells were grown to 70%–80% confluence, and then incubated with DMEM with 2% (v/v) heat-inactivated horse serum (Gibco) and 1% (w/v) P/S for 4–6 days. The medium was changed daily. The fully differentiated myotubes were used in the following experiments.
4
Primary Hippocampal Neuron Isolation from Rodents
5
Sheep Myoblast Isolation and Differentiation
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According to previous studies, sheep myoblasts were isolated by a two-step enzymatic method using muscle from newborn 5-day-old lambs [7 (link)]. Briefly, leg muscles were cut into small pieces and washed three times with DPBS, digested with 0.1% type I collagenase (Sigma-Aldrich, Saint Louis, MO, USA) for 1 h, and then digested with 0.25% trypsin (Gibco, Grand Island, NY, USA) for 30 min. The tubes were shaken every 10 min and filtered through a 200-mesh sieve. The cells were cultured in a growth medium consisting of DMEM-F12 (Gibco, Grand Island, NY, USA) supplemented with 20% FBS and 10% heat-inactivated horse serum (Gibco, Grand Island, NY, USA). Two hours later, the cell supernatant was transferred to a new flask and the cells began to adhere after 48 h. Myoblasts within four generations were used for subsequent studies. Differentiation of the myoblasts was carried out in a medium containing 2% horse serum in DMEM-F12. The differentiation was observed at 0, 72, and 120 h after differentiation.
6
Rat Pheochromocytoma Cell Culture
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Wild type PC12 and M-M17-26 (dominant negative H-Ras protein-expressing)12 (link) rat pheochromocytoma cells were cultured in DMEM containing 5% fetal calf serum and 10% heat-inactivated horse serum (GIBCO, Paisley, Scotland). Both cell lines were kindly provided by G.M. Cooper (Department of Biology, Boston University, MA).
7
Establishing PC-12 and Astrocyte Cell Cultures
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PC-12 cells were from the American Type Culture Collection (CRL-1721) and subjected to quality control tests by the American Type Culture Collection. PC-12 cells were cultured in Dulbecco's modified Eagle's medium (high glucose; Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 5% heat-inactivated horse serum (HyClone), penicillin (100 units/ml), and streptomycin (1 mg/ml) in plates coated with 10 μg/ml type IV collagen (Sigma–Aldrich). Cells were passaged no more than eight times.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-d -lysine-coated surfaces. Experiments were performed within 48 h of completing the isolation procedure.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-
8
Generating Ventricular-like hiPSC-CMs for Patch Clamp
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Ventricular-like hiPSC-CMs were differentiated from an in-house control hiPSC cell line [9 (link)]. Differentiated hiPSC-CMs were used to generate three-dimensional EHT (1 × 106 cells/100 μL EHT). The expansion and differentiation of hiPSC-CMs and the generation of EHT were performed according to published in-house standardized protocol [10 (link)]. EHT was cultured for 24–29 days under identical conditions at 37 °C in a 7% CO2 and 40% O2humidified cell culture incubator with a medium consisting of DMEM (Biochrom, Berlin, Germany), 10% heat-inactivated horse serum (Gibco, Paisley, Scotland), 1% penicillin–streptomycin (Gibco, Paisley, Scotland), insulin (10 μg/mL; Sigma, St. Louis, MO, USA), and aprotinin (33 μg/mL; Sigma, St. Louis, MO, USA). After culturing, hiPSC-CMs applied for patch clamp measurements were isolated with collagenase II (200 U/mL; Worthington Biochemical, Lakewood, NJ, USA) for 5 h. Isolated cells were plated on gelatine-coated (0.1%) glass coverslips (12 mm diameter; Carl Roth GmbH + Co, Karlsruhe, Germany) and kept in culture for 24–48 h to maintain adherence under superfusion in the recording chamber during patch clamp measurements.
9
Maintenance of TK6 Lymphoblastoid Cells
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Human lymphoblastoid TK6 cells were provided by Professor Kuicheng Zheng (Fujian Center for Disease Control and Prevention, China). The TK6 cells were maintained in RPMI 1640 medium (Gibco, USA) supplemented by 10% heat-inactivated horse serum (Gibco, USA) at 37°C under a humidified atmosphere and 5% CO2.
10
Culturing S. aureus with Supplements
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S. aureus strains were cultured in tryptic soy broth or tryptic soy agar at 37 °C, unless otherwise stated, and media were supplemented with 10 μg/ml chloramphenicol for plasmid selection and 0.2% heat-inactivated horse serum (Gibco/Life Technologies) to trigger secretion.
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