Heat inactivated fetal bovine serum hi fbs

Manufactured by Thermo Fisher Scientific

Sourced in United States

Heat-inactivated fetal bovine serum (HI-FBS) is a cell culture supplement derived from the blood of bovine fetuses. It is prepared by inactivating the serum through heat treatment, which helps to denature the proteins and inactivate any potential contaminants. HI-FBS is commonly used as a growth supplement in cell culture media to support the growth and proliferation of various cell lines.

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9 protocols using heat inactivated fetal bovine serum hi fbs

Inhibitor Preparation and Cell Culture Protocols

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PARPi (O, olaparib, AZD2281), ATRi (A, ceralasertib, AZD6738), and CHK1i (C, MK-8776) were purchased from Selleck Chemicals (Houston, TX, USA). The stock solutions of studied inhibitors were separately prepared from powders dissolved in 100% dimethyl sulfoxide (DMSO), aliquoted, and stored at −80 °C for up to a maximum of six months. RPMI 1640 and DMEM culture media, heat-inactivated fetal bovine serum (HI-FBS), and trypsin-EDTA were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Details concerning other key reagents used in the studies are included in Section 2 and Supplementary Tables (Tables S1–S3). Chemicals and solvents were obtained from Sigma-Aldrich (Saint Louis, MO, USA) or Avantor Performance Materials Poland S.A. (Gliwice, Poland).

Biegała Ł., Gajek A., Marczak A, & Rogalska A. (2023). Olaparib-Resistant BRCA2MUT Ovarian Cancer Cells with Restored BRCA2 Abrogate Olaparib-Induced DNA Damage and G2/M Arrest Controlled by the ATR/CHK1 Pathway for Survival. Cells, 12(7), 1038.

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Murine Breast Cancer Cell Culture

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The murine breast cancer cell line E0771 (CH3 Biosystems, Amherst, New York) was cultured in RPMI

1 \times 1640

with

300 mg / L

of L-glutamine (Gibco Gaithersburg, Maryland). 4T07 cells were acquired from American Type Culture Collection and cultured in DMEM with

4.5 g / L

of D-glucose,

584 mg / L

of L-glutamine, and

110 mg / L

sodium pyruvate (Gibco Gaithersburg, Maryland). Both cell media were supplemented with 10% v/v heat-inactivated fetal bovine serum (HI-FBS) (Gibco Gaithersburg, Maryland) and 1% v/v anti-mycotic/antibiotic (A/A) (Gibco, Gaithersburg, Maryland). Cells were maintained in a humidified 37°C incubator with 5%

{CO}_{2}

. Prior to the experiment, two black, 96-well plates with clear bottoms (Greiner, Bio-One, Kremsmünster, Austria) per cell line were plated with

4 \times 10^{4} cells / well

and incubated for 24 h. This resulted in near 100% confluence for each cell line at the time of the study.

Rickard A.G., Zhuang M., DeRosa C.A., Zhang X., Dewhirst M.W., Fraser C.L, & Palmer G.M. (2020). Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells. Journal of Biomedical Optics, 25(11), 116504.

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Culturing Cells with Exosome-Depleted Serum

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Iscove’s Modified Dulbecco’s Medium (IMDM), F12-K Nutrient Mixture Kaighn’s Modification with (+) L Glutamine, Dulbecco’s Modified Eagle Medium (DMEM)/F12 Nutrient Mixture without phenol red, penicillin/streptomycin (P/S), heat-inactivated fetal bovine serum (HI-FBS) and exosome-depleted fetal bovine serum (ED-FBS), which contains over 90% of exosomes depleted, were purchased from Gibco (Grand Island, NY). Phosphate buffered saline (PBS) and 0.25% trypsin-EDTA were from Invitrogen Life Technologies (Carlsbad, CA). Dimethyl sulfoxide (DMSO) was purchased from Tocris Bioscience (Bristol, United Kingdom). Forskolin and 2-mercaptoethanol were purchased from Sigma-Aldrich (St. Louis, MO). Mono-2-ethylhexyl phthalate (MEHP) was purchased from AccuStandard (New Haven, CT).

Harris S.M., Su A.L., Dou J.F., Loch-Caruso R., Elkin E.R., Jaber S, & Bakulski K.M. (2023). Placental Cell Conditioned Media Modifies Hematopoietic Stem Cell Transcriptome In Vitro. bioRxiv.

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Dynasore-mediated Mitochondrial Oxidative Stress

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Dynasore (minimum purity > 99%) (Cat# 2897) and mouse DuoSet kits for IL-1β/IL-1F2, TNF-α, IL-6, and IL-12 were purchased from R&D Systems (Minneapolis, MN, USA). The molecular structure of Dynasore is illustrated in

Supplementary Figure S1

. LPS from Escherichia coli O111:B4 (Cat# L4391) was purchased from Sigma-Aldrich (St Louis, MO, USA). The XTT Cell Viability Kit (Cat# 9095) was purchased from Cell Signaling Technology (Danvers, MA, USA). MitoSOX^TM Red mitochondrial superoxide indicator (Cat# M36008) was obtained from Thermo Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM) and heat-inactivated fetal bovine serum (HIFBS) were purchased from Gibco (Waltham, MA, USA). Recombinant murine colony-stimulating factor (M-CSF; Cat# 315–02) was purchased from PeproTech (Waltham, MA, USA).

Shan M., Wan H., Ran L., Ye J., Xie W., Lu J., Hu X., Deng S., Zhang W., Chen M., Wang F, & Guo Z. (2024). Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis. Drug Design, Development and Therapy, 18, 1369-1384.

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RAW 264.7 Murine Macrophage Cell Assay

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RAW 264.7 murine macrophage cells (KCLB No. 40071) were obtained from the Korea Cell Line Bank, Seoul, Korea. Dulbecco’s modified Eagle’s medium (DMEM), antibiotics and heat-inactivated fetal bovine serum (HI-FBS) were purchased from Life Technology (Rockville, USA). Lipopolysaccharide (E. coli, Serotype 0111:B4), and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits for TNFα, IL-6 and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies to iNOS, β-actin, NFκB, and Lamin B, and Horseradish peroxidase (HRP)-conjugated secondary antibodies were bought from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). P38, phospho-P38, JNK, phospho-JNK, ERK, phospho-ERK, STAT3, phospho-STAT3 (Tyr705), STAT1, and phospho-STAT1 (Tyr701) antibodies were procured from Cell Signaling Technology (Beverly, MA, USA). Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) was obtained from Abcam (Cambridge, MA).

Venkatesan T., Choi Y.W., Lee J, & Kim Y.K. (2017). Pinus densiflora needle supercritical fluid extract suppresses the expression of pro-inflammatory mediators iNOS, IL-6 and IL-1β, and activation of inflammatory STAT1 and STAT3 signaling proteins in bacterial lipopolysaccharide-challenged murine macrophages. DARU Journal of Pharmaceutical Sciences, 25, 18.

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Evaluating Cellular Responses to Oxidative Stress

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UM cells MEL270 HEK 293 cells and MCF-7 and RPMI-1640 medium (30–2001) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). Heat-inactivated fetal bovine serum (HI-FBS) (10438–026) and Penicillin-Streptomycin (15140122) were purchased from Life Technologies (Grand Island, NY). Anti-Rock1 antibody (rabbit-4035), anti-Diap1 antibody (rabbit-5486), and anti-p62 (rabbit-5114), anti-YAP1 (rabbit-4912), anti-β-actin antibody (rabbit-2128), anti-LC3A/B antibody was (rabbit-4108) and HRP-linked secondary antibodies- anti rabbit (7074S, 7076S) were obtained from Cell Signaling Technology (Danvers, MA), anti-TEF1 was purchased by Abcam (rabbit-ab133533). Methionine sulfoxide antibody cat #600160 was from Cayman, USA. N-acetyl-L-cysteine and L-histidine were purchased from Sigma-Aldrich (A7250 and H8000 respectively). Verteporfin (Visudyne) was obtained from Novartis (Novartis, Basel, Switzerland) and was dissolved following the manufacture’s protocol.

Konstantinou E.K., Notomi S., Kosmidou C., Brodowska K., Al-Moujahed A., Nicolaou F., Tsoka P., Gragoudas E., Miller J.W., Young L.H, & Vavvas D.G. (2017). Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity. Scientific Reports, 7, 46581.

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Isolation and Co-culture of Trigeminal Neurons

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Trigeminal ganglia were harvested from Nav1.8-DTA and control mice, and neurons were isolated via enzymatic digestion process, as previously described [26 (link), 27 (link)]. Single cell neuronal suspensions at the concentration of (~ 5 × 10⁴ neurons/insert) were seeded on 24-well plate polyethylene cell culture inserts (1 μm-pore sizes) (Millipore-Sigma) precoated with 50 μg/ml laminin (Sigma) in growth medium containing Alpha Modified Eagle’s Medium (αMEM) (Life Technologies), 10% heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies), and 1X Glutamine-Penicillin- Streptomycin (Invitrogen). Neurons were allowed to attach for 24 h in 5% CO₂ and 37 °C in the presence of 10 μM cytosine β-D- arabinofuranoside (Ara-C) (Sigma) to halt the non- neuronal cell proliferation [28 (link), 29 (link)]. The inserts containing the neuronal primary cultures were transferred to 24-well plates containing osteoblast precursor cells or induced RAW 264.7 cell lines for osteoblast and osteoclast co-cultures, respectively.

Austah O.N., Lillis K.V., Akopian A.N., Harris S.E., Grinceviciute R, & Diogenes A. (2022). Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis. Cellular and Molecular Life Sciences, 79(6), 330.

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Reagents for Cell Culture Experiments

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All chemicals used were of the highest purity available from Sigma-Aldrich (St. Louis, Mo) unless otherwise specified. Dulbecco’s PBS (no Ca²⁺, no Mg²⁺), Dulbecco’s Modified Eagle’s media (DMEM) and heat-inactivated fetal bovine serum (hi-FBS) were purchased from Life Technologies (Carlsbad, CA).

Guirado E., Rajaram M.V., Chawla A., Daigle J., La Perle K.M., Arnett E., Turner J, & Schlesinger L.S. (2018). Deletion of PPARγ in lung macrophages provides an immunoprotective response against M. tuberculosis infection in mice. Tuberculosis (Edinburgh, Scotland), 111, 170-177.

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Isolation of Trigeminal Ganglia Neurons

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Trigeminal ganglia were harvested from Nav1.8-DTA and control mice, and neurons were isolated via enzymatic digestion process, as previously described [26, 27] (link). Single cell neuronal suspensions at the concentration of (~ 5x10 4 neurons/insert) were seeded on 24-well plate polyethylene cell culture inserts (1µm-pore sizes) (Millipore-Sigma) precoated with 50µg/ml laminin (Sigma) in growth medium containing Alpha Modi ed Eagle's Medium (αMEM) (Life Technologies), 10% heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies), and 1X Glutamine-Penicillin-Streptomycin (Invitrogen).
Neurons were allowed to attach for 24 hours in 5% CO 2 and 37ºC in the presence of 10µM cytosine β-Darabinofuranoside (Ara-C) (Sigma) to halt the non-neuronal cell proliferation [28, 29] . The inserts containing the neuronal primary cultures were transferred to 24-well plates containing osteoblast precursor cells or induced RAW 264.7 cell lines for osteoblast and osteoclast co-cultures, respectively.

Austah O., Lillis K.V., Akopian A.N., Harris S.E., Simanavicious R., & Diogenes A. (2022). Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

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