Heat inactivated fcs

Manufactured by Thermo Fisher Scientific

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Heat-inactivated FCS is a sterile-filtered, heat-treated fetal calf serum that has been inactivated to reduce the level of complement and other heat-labile components. This product is suitable for use in cell culture applications where the presence of active complement or other heat-labile factors may interfere with experimental results.

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Heat-inactivated (129 citations) Heat-inactivated fetal calf serum (FCS; (7 citations) Heat-inactivated foetal calf serum (FCS; (2 citations)

36 protocols using heat inactivated fcs

Naive CD4+ T Cell Activation and Polarization

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Naive CD4⁺ T cells were labelled by Cell Trace Violet (ThermoFisher Scientific) according to the manufacturer’s protocol and activated in vitro using a combination of plate-bound anti-CD3 (2 μg/mL) (BioLegend) and soluble anti-CD28 (2 μg/mL) (BioLegend) antibodies in RPMI (Merck), containing 10% Heat Inactivated FCS (Invitrogen) and 1% Penicillin-Streptomycin (Merck). Cell proliferation was measured as dilution of the cell dye as assessed by flow cytometry.
For in vitro polarization, naive T cells were freshly isolated and subsequently cultured for 4/5 days in RPMI (Merck), containing 10% Heat Inactivated FCS (Invitrogen) and 1% Penicillin-Streptomycin (Merck). Following conditions were used; Th1: 50 U/mL IL-2, 1 μg/mL anti-CD28, 1 μg/mL anti-CD3, 3.5 ng/mL IL-12, 10 μg/mL anti-IL4; Th2: 50 U/mL IL-2, 1 μg/mL anti-CD28, 1 μg/mL anti-CD3, 10 μg/mL IL-4, 10 μg/mL anti-IFN-γ; Th17: 1 µg/mL anti-CD28, 5 ng/mL TGFβ, 10 ng/mL IL-1b, 50 ng/mL IL-6, 20 ng/mL IL-23, 10 µg/mL anti-IFN-γ, 10 µg/mL anti-IL-4; Tregs: 50 U/mL IL-2, 1 µg/mL anti-CD28, 5 ng/mL TGFβ.

Oftedal B.E., Maio S., Handel A.E., White M.P., Howie D., Davis S., Prevot N., Rota I.A., Deadman M.E., Kessler B.M., Fischer R., Trede N.S., Sezgin E., Maizels R.M, & Holländer G.A. (2021). The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function. Communications Biology, 4, 681.

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Differentiation and Stimulation of THP-1 Macrophages

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Human acute monocytic leukemia cells (THP-1) were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and were maintained in RPMI 1640 media (ThermoFisher Scientific, UK) supplemented with 10% heat-inactivated FCS (ThermoFisher Scientific, UK) and 1% Penicillin/Streptomycin (ThermoFisher Scientific, UK). For differentiation, phorbol 12-myristate 13-acetate (100 ng/ml; Sigma-Aldrich, Dorset, UK) was added to THP-1 cell suspension and cells were incubated for 72 hr period followed by 24 hr rest in fresh media. For LPS stimulation experiments, THP-1 macrophages were stimulated with P. aeruginosa LPS alone (100 ng/ml; Serotype 10, Source strain ATCC 27316; Sigma-Aldrich) or LPS in combination with peptide and incubated for 16 hr. Supernatants were collected for analysis by ELISA. For cell viability assays, THP-1 macrophages were incubated with Fluorofire-Blue ProViaTox kit (Molecutools, Dublin, Ireland) as per manufacturer’s instructions. Staurosporine (Sigma Aldrich, 10 mM) was used as positive control.

Carlile S.R., Shiels J., Kerrigan L., Delaney R., Megaw J., Gilmore B.F., Weldon S., Dalton J.P, & Taggart C.C. (2019). Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection. Scientific Reports, 9, 6071.

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Generation of SARS-CoV-2-Reactive CD8+ T Cell Lines

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CD8+ T cell lines were generated as previously described [26 (link),30 (link)]. In summary, HLA typed HLA-A*02:01+ PBMCs from COVID-19 recovered individuals were incubated with SARS-CoV-2 peptide pools (2 μM/peptide) and cultured for 10–14 days in RPMI-1640 supplemented with 1× Non-essential amino acids (NEAA; Thermofisher, Scoresby, Australia), 5 mM HEPES (Thermofisher, Scoresby, Australia), 2 mM L-glutamine (Thermofisher, Scoresby, Australia), 1× penicillin/streptomycin/glutamine (Thermofisher, Scoresby, Australia), 50 µM 2-ME (Sigma-Aldrich, St Louis, MO, USA) and 10% heat-inactivated FCS (Thermofisher). The cultures were supplemented with 10 IU IL-2 (BD Biosciences, Melbourne, Australia) 2–3 times weekly. CD8+ T cell lines were freshly harvested and used for subsequent assays.

Szeto C., Nguyen A.T., Lobos C.A., Chatzileontiadou D.S., Jayasinghe D., Grant E.J., Riboldi-Tunnicliffe A., Smith C, & Gras S. (2021). Molecular Basis of a Dominant SARS-CoV-2 Spike-Derived Epitope Presented by HLA-A*02:01 Recognised by a Public TCR. Cells, 10(10), 2646.

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Inducing N2a Cell Differentiation

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Undifferentiated N2a cells were cultured in 24‐well plates with DMEM (Thermo Fisher, Chelmsford, MA, USA) containing penicillin/streptomycin (Thermo Fisher) and 10% heat‐inactivated FCS (Thermo Fisher). To induce differentiation, N2a cells were treated with DMEM containing 0.1% serum and 5 μmol/L RA (Sigma), together with the indicated natural compounds, for 24 hours. Differentiated N2a cells were stained with Neurite Outgrowth Staining Kit (Thermo Fisher) and imagined using an inverted microscope (Eclipse Ti‐E Inverted Microscope, Nikon, Japan). For each treatment group, about 200 randomly selected cells were counted to obtain the proportion of neurite‐bearing cells.

Chen L., Feng P., Peng A., Qiu X., Lai W., Zhang L, & Li W. (2020). Protective effects of isoquercitrin on streptozotocin‐induced neurotoxicity. Journal of Cellular and Molecular Medicine, 24(18), 10458-10467.

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Culturing HLA-A*0201-restricted CD8+ T-cell Clones

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The ILA1 CD8⁺ T-cell clone is specific for the HLA-A*0201-restricted human telomerase reverse transcriptase-derived epitope ILAKFLHWL (residues 540–548) (39 (link)), and the 1E6 T-cell clone is specific for the human leukocyte antigen HLA-A*0201-restricted autoantigen preproinsulin epitope ALWGPDPAAA (residues 15–24) (40 (link)). CD8⁺ T-cell clones were maintained in RPMI 1640 medium (Life Technologies) containing 100 units/ml penicillin (Life Technologies), 100 mg/ml streptomycin (Life Technologies), 2 mml-glutamine (Life Technologies), and 10% heat-inactivated FCS (Life Technologies) (R10) supplemented with 2.5% Cellkines (Helvetica Healthcare, Geneva, Switzerland), 200 IU/ml IL-2 (PeproTech, Rocky Hill, NJ), and 25 ng/ml IL-15 (PeproTech). Hmy.2 C1R B-cells expressing full-length HLA-A*0201 were generated as described previously (41 (link)).

Cole D.K., van den Berg H.A., Lloyd A., Crowther M.D., Beck K., Ekeruche-Makinde J., Miles J.J., Bulek A.M., Dolton G., Schauenburg A.J., Wall A., Fuller A., Clement M., Laugel B., Rizkallah P.J., Wooldridge L, & Sewell A.K. (2016). Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase. The Journal of Biological Chemistry, 292(3), 802-813.

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BM Cell Culture with Immunosuppressants

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BM cells were cultured in IMDM (containing 200 ng/ml mrFlt3-L (Stem cells), 10% heat-inactivated FCS (Life Technologies), streptomycin 100 mg/ml, and penicillin 100 U/ml) at 3 × 10⁶cells per milliliter. For culture of sorted progenitors, stem cell factor (SCF; 50 ng/ml), IL-6 (20 ng/ml), and IL-3 (10 ng/ml; all R&D Systems, Minneapolis, MN, http://www.rndsystems.com) were added (referred to as HSC medium). Two micrograms per milliliter CsA or 0.2 µg/ml FK506 (Cell Signaling Technology, Danvers, MA, www.cellsignal.com) were added to the cell culture for 30 minutes preceding the addition of Flt3-L/GM-CSF and were maintained throughout the culture.

Fric J., Lim C.X., Mertes A., Lee B.T., Viganò E., Chen J., Zolezzi F., Poidinger M., Larbi A., Strobl H., Zelante T, & Ricciardi-Castagnoli P. (2014). Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L. Stem Cells (Dayton, Ohio), 32(12), 3232-3244.

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Purification of GFP-tagged Synapsin I

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The expression vector for GFP-tagged rat SynI (a-isoform; GFP-SynI) was kindly donated by H.-T. Kao (Brown University, Providence, RI). GFP-Syn I was expressed in HEK293T cells using calcium phosphate (40 μg GFP-SynI for 3.5 × 10⁶ cells/150 mm dish). HEK293T cells were routinely cultured in IMDM (Sigma-Aldrich), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, glutamine, and 10% heat-inactivated FCS (Life Technologies). HEK293T cells were lysed in buffer that contained 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.5 mM Tris-2-carboxyethyl-phosphine hydrochloride (TCEP), and protease inhibitors (1 mM PMSF/1 mM pepstatin; Sigma-Aldrich). The lysate was centrifuged for 1 h at 17,000 g, followed by affinity purification of GFP-SynI using GFP-Trap A (Chromotek, Germany) for 2 h. After extensive washes in washing buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM TCEP), bound proteins were eluted by 250 μL of 0.2 M glycine pH 2.5 followed by 30 s incubation under constant mixing and centrifugation. The supernatant was immediately neutralized by adding 25 μL of Tris-base (pH 10.4) and stored at −80 °C until use.

Rocchi A., Sacchetti S., De Fusco A., Giovedi S., Parisi B., Cesca F., Höltje M., Ruprecht K., Ahnert-Hilger G, & Benfenati F. (2019). Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function. Cell Death & Disease, 10(11), 864.

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Th Cell Differentiation Assay

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Sorted Pik3cd GOF or WT naïve CD4⁺ T cells were cultured in flat bottom 96 well plates previously coated overnight with anti-CD3 (BioLegend) (4 μg/mL) in RPMI1640 (Life technologies) supplemented with 10% heat inactivated FCS (Life technologies), 5×10⁻⁵ M 2-ME, 0.1mM non essential amino acids, 1mM sodium pyruvate, 10mM HEPES, 100u/mL penicillin, 100ug/mL Streptomycin, 100ug/mL Noromycin (all from Sigma) at a density of 0.5 ×10⁶ cells/mL. CD4⁺ naïve cells were cultured for 4 days in the following conditions: Th0 - anti-CD28 1 μg/mL + anti-TGFβ 5 μg/mL + anti-IL-4 5 μg/mL + anti-IFNγ 5 μg/mL; Th1 - anti-CD28 1 μg/mL + anti-TGFβ 5 μg/mL + anti-IL-4 5 μg/mL + IL-12 10ng/mL; Th2 - IL-4 10ng/mL + anti-CD28 1 μg/mL + anti-TGFβ 5 μg/mL + anti-IFNγ 5 μg/m; Th17 - IL-6 20ng/mL + human TGFβ 1ng/mL + anti-IFNγ 5 μg/m + anti-IL-4 5 μg/mL + anti-CD28 1 μg/mL. After 4 days of culture, cells were stimulated with PMA (50ng/mL) and ionomycin (375ng/mL) for 6 hours. Brefeldin A (10 μg/mL) was added to each well at 2 hours of culture. Cells were then harvested, washed and stained with Zombie Aqua Viability dye (BioLegend). CD4⁺ T cells were fixed with 2% formalin, permeabilised with saponin (0.1%), and stained intracellularly with mAbs directed against TNFα, IFNγ, IL17A, IL-2, IL5, IL-4 and analysed by flow cytometry.

Bier J., Rao G., Payne K., Brigden H., French E., Pelham S.J., Lau A., Lenthall H., Edwards E.S., Smart J.M., Cole T.S., Choo S., Joshi A.Y., Abraham R.S., O’Sullivan M., Boztug K., Meyts I., Gray P.E., Berglund L.J., Hsu P., Wong M., Holland S.M., Notarangelo L.D., Uzel G., Ma C.S., Brink R., Tangye S.G, & Deenick E.K. (2019). Activating mutations in PIK3CD and murine CD4+ T cells. The Journal of allergy and clinical immunology, 144(1), 236-253.

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Profiling Follicular B Cell Responses

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Spleens of Pik3cd GOF, Pik3r1 LOF, and WT mice were harvested, and single-cell suspensions were prepared. Splenocytes were labeled with mAb against B220, CD21/35, CD23, and CD93, and follicular B cells (B220⁺ CD23⁺ CD21^int CD93⁻) were sorted using a FACSAria III (Beckton Dickinson). Purity of the recovered populations was typically >98%. Sorted follicular B cells were labeled with CellTrace Yellow and then cultured in 48-well plates in RPMI1640 (Life Technologies) supplemented with 10% heat-inactivated FCS (Life Technologies), 5 × 10⁻⁵ M 2-ME, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 100 U/ml penicillin, 100 µg/ml Streptomycin, 100 µg/ml Noromycin (all from Sigma-Aldrich) at a density of 0.2 × 10⁶ cells/ml. B cells were stimulated with 10 µg/ml anti-CD40 clone FGK4.5 (Enzo Live Sciences) alone or in combination with 10 ng/ml IL-4 (R & D Systems), or 10 µg/ml LPS (Sigma-Aldrich) alone or together with 0.5 ng/ml human TGFβ (Peprotec). After 4 d, cells were harvested, washed, and stained with Zombie Aqua Viability Dye (BioLegend). Cells were then surface stained with B220, fixed with 2% formaldehyde, and permeabilized with saponin. Permeabilized cells were stained with mAbs to IgG1, IgE, IgG2b, and IgG3, and analyzed by flow cytometry.

Nguyen T., Lau A., Bier J., Cooke K.C., Lenthall H., Ruiz-Diaz S., Avery D.T., Brigden H., Zahra D., Sewell W.A., Droney L., Okada S., Asano T., Abolhassani H., Chavoshzadeh Z., Abraham R.S., Rajapakse N., Klee E.W., Church J.A., Williams A., Wong M., Burkhart C., Uzel G., Croucher D.R., James D.E., Ma C.S., Brink R., Tangye S.G, & Deenick E.K. (2023). Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2. The Journal of Experimental Medicine, 220(6), e20221020.

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Differentiation of Murine Bone Marrow-Derived Macrophages

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Bone marrow was harvested from 6-week-old C57BL/6 mice and cultured at 37°C/5% CO₂ in RPMI 1640 Medium (Gibco) containing 10% v/v heat-inactivated FCS (Gibco), 2-mercaptoethanol (Sigma-Aldrich) and 5% penicillin/streptavidin (Life Technology). The cells were supplemented with 50ng/ml recombinant macrophage colony stimulating factor (Miltenyi Biotec) on days 1 and 3 of culture to stimulate the differentiation of monocytes to macrophages. At day 6, differentiated BMDMs were harvested and used for experiments. Culture and stimulation conditions for each experiment are described in the respective figure legends. Ethical approval for these studies was granted by the University of Technology Sydney (UTS) Animal Care and Ethics Committee (Approval Number: ETH18-2257) and experiments were conducted in accordance with the approved guidelines to be compliant with The Australian Code for the Care and Use of Animals for Scientific Purposes.

Quinteros S.L., von Krusenstiern E., Snyder N.W., Tanaka A., O’Brien B, & Donnelly S. (2023). The helminth derived peptide FhHDM-1 redirects macrophage metabolism towards glutaminolysis to regulate the pro-inflammatory response. Frontiers in Immunology, 14, 1018076.

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