Spleens were aseptically obtained from immunized and control mice, macerated using a sterile tissue culture filter (BD Biosciences, San Jose, CA), and the resulting cell suspension was diluted in Hanks Balanced Salts Solution (Invitrogen, Carlsbad, CA) containing 10% Heat Inactivated Fetal Calf Serum (Invitrogen, Carlsbad, CA). Contaminating red blood cells were lysed using the Mouse Erythrocyte Lysing Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s directions. Cells were then harvested by centrifugation and diluted into R10 media (RPMI media (Invitrogen, Carlsbad, CA), supplemented with 10% Heat Inactivated Fetal Calf Serum (Invitrogen, Carlsbad, CA), 10 mM HEPES (Invitrogen, Carlsbad, CA), 50 μM 2-mercaptoethanol (Invitrogen, Carlsbad, CA), and 100 μg/mL each of Penicillin G and Streptomycin (Invitrogen, Carlsbad, CA). Cell viability counts were performed on remaining splenocytes using trypan blue exclusion staining with the aid of a light microscope (Fisher Micromaster Inverted, Fisher Scientific, Pittsburgh, PA) and an Improved Nebauer hemacytometer (Hausser Scientific, Horsham, PA). Cells were then diluted using R10 to concentrations ranging from 0.64–4.0 x 106 viable cells/mL.
Heat inactivated
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Japan
Heat-inactivated is a laboratory equipment product used for thermal inactivation of biological samples. It provides a controlled environment to expose samples to heat, effectively deactivating or destroying specific biological components within the samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (90 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (42 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (24 mentions)
L glutamine, by Thermo Fisher Scientific (20 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (15 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (14 mentions)
Penicillin, by Thermo Fisher Scientific (14 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (9 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (8 mentions)
Rpmi 1640, by Thermo Fisher Scientific (8 mentions)
Anti cd34 coated magnetic beads, by Miltenyi Biotec (7 mentions)
Tnf α, by R&D Systems (7 mentions)
Rpmi 1640, by Eurobio Scientific (7 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (7 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Horse serum, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Bovine serum, by Thermo Fisher Scientific (3 mentions)
129 protocols using heat inactivated
1
Splenic Cell Isolation and Characterization
2
Human PBMC Isolation and Stimulation
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After approval of our experimental protocol by our institution committees (Institut Pasteur de Lille, agreement N° DC 2013-2022) in accordance with relevant guidelines and regulations, blood samples were collected from five healthy donors, after signed agreement. Peripheral blood mononuclear cells (PBMCs) were isolated after Ficoll gradient centrifugation (GE Healthcare Bio-Sciences, Uppsala, Sweden), as described before [51 (link)]. Cells were washed and adjusted to 2 × 106 cells/mL in RPMI 1640 (Gibco, Life Technologies, Ghent, Belgium) supplemented with 150 μg/mL gentamicin, 2 mM glutamine and 10% heat-inactivated FCS (Gibco, Life Technologies, Grand Island, NE, USA). PBMCs were stimulated with PBS or bacteria (ratio cells/bacteria of 1:10) four 24 h at 37 °C under 5% CO2. Supernatants were collected, clarified by centrifugation and stored at −20 °C. Cytokine measurements were performed using R&D Duoset ELISA kits (R&D, Minneapolis, MN, USA).
3
RAW 264.7 Cell Culture Protocol
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RAW 264.7 cells were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco, Langley, OK, USA) supplemented with 10% heatinactivated fetal calf serum (Gibco), 50 IU/mL penicillin, and 100 mg/mL streptomycin. Cells were subcultured every 3 to 4 days and seeded at 106/mL in six-well plates (Nalge Nunc, Rochester, NY, USA).
4
Mobilization and Isolation of CD34+ HSC
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Example 29
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
5
Cell Culture Conditions for Diverse Viral Strains
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PDCoV strain CH-HA3-2017, PSV strain JXXY-a2, PRV strain DX, PCV2 strain HZ0201, PTV, and SeV were stored in our lab (74 (link)– (link)77 (link)). ST cells (ATCC CRL-1746) were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, USA) supplemented with 8% heat-inactivated fetal bovine serum (FBS; Gibco, USA). LLC-PK cells (ATCC CL-101) were cultured at 37°C in 5% CO2 in minimum essential medium (MEM; HyClone, USA) supplemented with 8% heat-inactivated FBS (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% HEPES buffer solution (Gibco, USA), and 1% MEM nonessential amino acids (NEAA; Gibco, USA). IPEC-J2 cells, a generous gift from Yaowei Huang at Zhejiang University, China, were cultured at 37°C in 5% CO2 supplemented with 8% heat-inactivated FBS (Biological Industries [BI], USA). PK-15 cells (ATCC CCL-33) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 5% heat-inactivated FBS (Gibco, USA).
6
Drosophila S2R+ Cell Culture Protocol
7
Differentiation of PC12 Cells with NGF
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PC12 cell line was obtained from Pasteur Institute of Iran (Tehran, Iran), and cultured on poly‐l‐ornithine (Sigma, USA) and laminin (Sigma)‐coated dishes in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% (v/v) heat‐inactivated horse serum (Sigma), 5% (v/v) heat‐inactivated fetal bovine serum (Gibco), and 100 U/ml penicillin–streptomycin (Gibco) at 37°C under a humidified atmosphere of 5% CO2. To induce differentiation, cells were treated for 7 days in medium containing 50 ng/ml of NGF‐β (Cell Guidance Systems, USA), 100 U/ml penicillin/streptomycin and 1% (v/v) horse serum. The half volume of differentiating medium was refreshed every 2 days.
8
Generating Bone Marrow-Derived Dendritic Cells
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BMDCs were differentiated by culture of Mst1+/+ and Mst1−/− mouse BM cells in the presence of 20 ng/ml GM-CSF for 8 days (9 (link)). Briefly, BM cells were isolated from the femur and tibia bones of Mst1+/+ and Mst1−/− 8- to 10-weeks old mouse (female), followed by RBC lysis. The BM cells were then cultured at a concentration of 2 × 105 cells per ml. The cells were cultured in RPMI containing 10% heat-inactivated FBS (Gibco), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin (Corning), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). The cells were supplemented with 20 ng/ml GM-CSF after 3, 5, and 7 days in the course of the 8-days culture or after 3, 5, 7, and 8 days in the course of the 10-days culture.
9
Folate Deficiency Impacts Adipocyte Differentiation
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Murine 3T3-L1 cells were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan, ROC). Cells were seeded on the 6-well-plate at 1 × 105 cells/well and cultured in Dulbecco's modified eagle medium (DMEM; Himedia, Mumbai, India) supplemented with 10% heat-inactivated bovine serum (Gibco, ThermoFisher, Grand Island, NY, USA). When reaching full confluence, cells were cultured with the differentiation medium composed of 0.2 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/ml insulin in 10% heat-inactivated fetal bovine serum (Gibco, ThermoFisher) DMEM. This differentiation medium was removed after 4 days and replaced with folic acid-free DMEM (Himedia) plus 10 μg/ml insulin either containing 0 μM (f0), 9.1 μM (f1), 45.3 μM (f5), or 90.6 μM (f10) folic acid. The medium was replenished every 2 days until adipocytes had acquired intracellular lipid droplets after 12-day incubation. For the proinflammatory cytokines experiment, preadipocytes were seeded on the 24-well-plate at 1.5×104 cells/well-cultured with f0 or f1 medium, and the supernatant was collected for cytokines assay after 4 days. A number of four to five independent experiments were performed for the in vitro folate deficiency study.
10
Mobilization and Expansion of CD34+ Stem Cells
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Example 38
After obtaining informed consent, healthy CD34+ stem cell donors receive rhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days for peripheral blood stem cell mobilization and then undergo apheresis for 2 consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC) are isolated from mobilized peripheral blood by Ficoll density gradient centrifugation and are split in two parts. One part is used to purify CD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec, Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+ fractions is controlled. CD34+-enriched HSC are then used immediately in the two-step culture method or frozen until use in the one-step culture method.
Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplemented with 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco, Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM (Gibco), supplemented with 10% heat-inactivated FBS, is used for expansion. Recombinant human stem cell factor (rhSCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, and TNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).
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