PBMCs were suspended at a density of 2 × 106 cells/mL in complete culture medium (RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamine, and 10% heat-inactivated fetal calf serum (Gibco, Waltham, MA, USA) in a 6-well plate. Cells were stimulated with 25 ng/mL phorbol myristate acetate (PMA) plus ionomycin (1 µg/mL) (Biovision, Milpitas, CA, USA) in the presence of brefeldin A (BioLegend, San Diego, CA, USA). Cells were incubated at 37 °C in presence of 5% CO2 for 6 h, then analyzed by flow cytometer.
Heat inactivated fetal calf serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, France, Austria
Heat-inactivated fetal calf serum is a cell culture reagent derived from the blood of fetal calves. It is processed by heat inactivation to deactivate any potential contaminants.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (23 mentions)
Penicillin, by Thermo Fisher Scientific (22 mentions)
L glutamine, by Thermo Fisher Scientific (13 mentions)
Streptomycin, by Merck Group (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Rpmi 1640, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Merck Group (10 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions)
L glutamine, by Merck Group (6 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium, by Merck Group (4 mentions)
Rpmi 1640, by Lonza (4 mentions)
Penicillin, by GE Healthcare (4 mentions)
Streptomycin, by GE Healthcare (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Penicillin g streptomycin, by Merck Group (2 mentions)
99 protocols using heat inactivated fetal calf serum
1
Stimulation and Analysis of PBMCs
2
Oxidative Stress and Autophagy in Renal Cells
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Normal rat kidney (NRK)-52E cells (a renal proximal tubular cell line) obtained from the American Type Culture Collection (Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, NY) supplemented with 50 IU/mL penicillin and 10% heat-inactivated fetal calf serum (Gibco Laboratories, Grand Island, NY) [15 (link)]. For experiments involving H2O2, 200, 400, or 600 μM H2O2 was added to the NRK-52E cells for 4 h. For starvation experiments, NRK-52E cells were incubated in HANKS medium without serum for 24 h. Expression vectors encoding wild-type human HSPB1 were obtained from OriGene Technologies, Inc. (Rockville, MD) and transfected into the NRK-52E cells via electroporation (360V, 960 μFD), as previously described [15 (link)]. Small interfering RNAs (siRNAs) specific for HSPB1 and LC3 and control-scrambled siRNAs were purchased from Life Technologies (Gaithersburg, MD). NRK-52E cells were transfected with the siRNAs via lipofection, as previously described [15 (link)]. Rapamycin was obtained from Focus Biomolecules (Plymouth meeting, PA) and bafilomycin A was obtained from LC Laboratories (Woburn, MA). H2O2 was obtained from Sigma-Aldrich Japan K.K. (Tokyo, Japan). All other chemicals were purchased from Funakoshi (Tokyo, Japan).
3
Murine and Hamster Cell Culture
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The murine mastocytoma cell line (P815, ATCC: TIB64) and the kidney carcinoma cell lines of hamsters (BSR, ATCC: CCL10) were kindly donated by the laboratory of Dr. Michel Lepoivre, 841 Institute of Biochemistry, University of Paris XI, France. The cell lines were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% Heat-inactivated fetal calf serum (Gibco BRL, Cergy Pontoise, France), penicillin G- streptomycin (1%), and 0.2% sodium bicarbonate (Sigma) at 37°C in a humidified atmosphere containing 5% CO2.
4
Bone Marrow-Derived Dendritic Cell Protocol
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Bone marrow-derived DCs were generated using methods described by Inaba et al. (1992) (link) and Lutz et al. (1999) (link). Briefly, BALB/c mice tibiae were removed and left in 70% ethanol for 2–5 min for disinfection and then washed in PBS. Cells within the marrow were disintegrated by vigorous pipetting. Thereafter, cells were cultured in medium RPMI-1640 (GIBCO, Germany) supplemented with Penicillin (100 U/ml, Sigma, Germany), Streptomycin (100 μg/ml, Sigma, Germany), L -glutamin (2 mM, Sigma, Germany), 10% heat-inactivated fetal calf serum (GIBCO, Germany), recombinant murine granulocyte–macrophage colony-stimulating factor (200 U/ml, perprotech, United States), recombinant murine interleukin-4 (5 ng/ml, perprotech, United States). At days 3, 5, 7 and 9, half of the cells culture supernatant was collected and centrifuged, the cell pellet resuspended and given back to the original plate, and then adding the equivalent of the medium. At day 10, cells can be used (BMDCs). The prepared BMDCs were treated with SEA (40 μg/ml) or untreated; after incubation for 24 h, the culture supernatant was harvested. Exosomes were purified from the supernatant using an exosome extraction kit (Invitrogen, United States) according to manufacturer’s instructions.
5
Murine TNBC and ER+ Cell Culture Protocol
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The 4T1 and TS/A spontaneous murine cells are TNBC and ER+ phenotypic lines, respectively, originated in BALB/C mice [37 (link),38 (link),39 (link),40 (link)]. The 4T1 (ATCC) cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units of penicillin, and 100 μg/mL of streptomycin. TS/A cells (Sigma-Aldrich Inc., St. Louis, MO, USA) were grown in Dulbecco’s modified MEM (GIBCO, Life Technologies, Monza, MB, Italy) supplemented with 2 mM of glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat-inactivated fetal calf serum (GIBCO, Life Technologies, Monza, MB, Italy) at 37 °C in humidified 5% of CO2 atmosphere. Culture medium was changed every 2–3 days, and cells were passaged with 0.25% trypsin/EDTA. Cells were routinely tested for Mycoplasma using a MycoAlert mycoplasma detection kit (BioWhittaker-Lonza, Euroclone S.p.a., Milan, Italy).
6
Quantification of HTLV-1 Proviral Load
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Specimens: In total five clinical samples were provided by a biomaterial bank of HTLV-1 carriers, JSPFAD [13 ,49 ]. The clinical samples were a part of those collected with an informed consent as a collaborative project of JSPFAD. The project was approved by the Institute of Medical Sciences, the University of Tokyo (IMSUT) Human Genome Research Ethics Committee. Information about the disease status of samples was obtained from JSPFAD database in which HTLV-1-infected individuals were diagnosed based on the Shimoyama criteria [50 (link)]. In brief, genomic DNA from PBMCs was isolated using a QIAGEN Blood kit. PVLs were measured by real-time PCR using the ABI PRISM 7000 Sequence Detection System as described in [10 (link)].
Cell lines: An IL2-dependent TL-Om1 cell line [51 (link)] was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 1% penicillin-streptomycin (GIBCO), and 10 ng/mL IL2 (R&D systems). The same conditions as those of patient samples were used to extract DNA and measure PVL.
Cell lines: An IL2-dependent TL-Om1 cell line [51 (link)] was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 1% penicillin-streptomycin (GIBCO), and 10 ng/mL IL2 (R&D systems). The same conditions as those of patient samples were used to extract DNA and measure PVL.
7
HL60 cell culture protocol
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HL60 undifferentiated promyelocytic leukemia cells, a validated cellular model for the study of different blood cell types as human neutrophils [14 (link)], were cultured in 25 cm2 flasks containing RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, ThermoFisher Scientific) at 37 °C with 5% CO2, as previously described [15 (link)].
8
Isolation and Activation of Naive CD4+ T Cells
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CD4+ T lymphocytes were isolated from splenocytes using cell negative isolation kits according to the manufacturer’s instructions (Invitrogen Dynal AS, Oslo, Norway). Twenty-four-well cell culture plates (Corning, Life Sciences, Amsterdam, Netherlands) previously coated with 2.5 μg mL−1 of anti-CD3 (clone 145-2C11) and 2.5 μg mL−1 of anti-CD28 (clone 37.51) monoclonal antibodies (mAbs) (BD Biosciences, San Jose, CA) were seeded with 5 × 105 purified naive CD4+ T cells (more than 92 % pure) in RPMI-1640 medium (GIBCO Life Technologies, Paisley, UK) supplemented with 10 % heat inactivated fetal calf serum (GIBCO Life Technologies), 1 % non-essential amino acids, 4-mM l -glutamine, 1-mM sodium pyruvate, 100-IU/ml penicillin, 100-μg/ml streptomycin (GIBCO-BRL), 10-mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 2 × 10-5 M 2-β-Mercapto-ethanol.
9
PBMC Isolation and Culture
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Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia, Uppsala, Sweden). The growth medium was supplemented with 10% heat-inactivated fetal calf serum (GIBCO, USA), 100 units/mL of penicillin and 100 μg/mL of streptomycin, and the cells were cultured at 37 °C with 5% CO2.
10
Caco-2 Cell Adhesion Assay for LAB
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The Caco-2 cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s Modified Eagle’s minimal essential medium (DMEM; Gibco) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Gibco), 2 mM l-glutamine (Sigma),100Uml−1 penicillin and 100 mg ml−1 streptomycin (Sigma) at 37 °C in 5% CO2 condition. For the adhesion assay, Caco-2 monolayers were seeded at a concentration of 5 × 105 cells per well in 12-well plates. Cells were treated with LAB suspension (1 × 10 7CFU/mL) for 2 h when the cells reached 80% confluence. After incubation, the adherent LAB bacteria and Caco-2 cells were detached by adding 1ml of 0.05% (v/v) Triton-X 100, and the CFU of adherent bacteria was calculated by serial dilution and spreading on MRS agar plates. The percentage of LAB adherence was calculated as follows:
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