Calf serum

Manufactured by Cytiva

Sourced in United States

Calf serum is a commonly used supplement in cell culture media. It provides a complex mixture of nutrients, growth factors, and other components that support the growth and proliferation of cells in vitro.

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Lab products found in correlation

20 protocols using calf serum

Maintenance of Cell Lines in DMEM

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Transformed African green monkey kidney fibroblast COS-7 cells (RCB0539, Riken BioResource Research Center, Tsukuba, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako, Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Biofluids, Fleming Island, FL) and nonessential amino acids (FUJIFILM Wako). Human breast cancer MCF-7 cells (Health Science Research Resources Bank, Osaka, Japan) were maintained in DMEM supplemented with 15% (v/v) FBS. A knockout cell line for human GDE7 gene (GDPD3) was previously established^{18 (link)}. 3T3-L1 cells (Japanese Collection of Research Bioresources Cell Bank, Ibaraki, Japan) were maintained in DMEM (1 g L⁻¹ glucose) supplemented with 10% (v/v) calf serum (Cytiva, Marlborough, MA). These cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.

Kitakaze K., Ali H., Kimoto R., Takenouchi Y., Ishimaru H., Yamashita A., Ueda N., Tanaka T., Okamoto Y, & Tsuboi K. (2023). GDE7 produces cyclic phosphatidic acid in the ER lumen functioning as a lysophospholipid mediator. Communications Biology, 6, 524.

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Osteocyte-like Cell Culture Protocol

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Murine Osteocyte-like cell line MLO-Y4 (Kerafast, Inc. Boston, MA) were cultured according to the suppliers recommended protocol.[76 (link)] The cell culture flasks were coated with 4μg/cm² rat tail type I collagen (Sigma-Aldrich, Inc. St. Louis, MO) for 30 minutes at 37°C before cell seeding. Alpha-Minimum Essential Media (Thermo-Fisher Scientific, Grand Island, NY) supplemented with 2.5% fetal bovine serum (FBS, R&D Systems, Minneapolis, MN), 2.5% calf serum (Cytiva Life Sciences, Marlborough, MA) and 1% penicillin/streptomycin (Thermo-Fisher Scientific) were used as cell culture media. The cells were seeded on collagen coated glass slides (Fisherbrand, Thermo-Fisher Scientific) at a density of 10,000 cells/cm² and cultured for a period of 1 to 3 days. Human osteoblast like Saos2 osteosarcoma cells (ATCC, Manassas, VA) were cultured with Dulbecco’s modification of eagle’s media (DMEM, Thermo-Fisher Scientific supplemented with 10% FBS (R&D Systems), 1%penicillin/streptomycin (Thermo-Fisher Scientific) and 1% Glutamax (Thermo-Fisher Scientific) using standard cell culture protocol.

Lovenberg T.W., Liaw C.W., Grigoriadis D.E., Clevenger W., Chalmers D.T., De Souza E.B, & Oltersdorf T. (1995). Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.. Proceedings of the National Academy of Sciences of the United States of America, 92(3), 836-840.

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Knockdown and Rescue of CFIm Subunits in Cell Lines

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NIH3T3 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum (Cytiva). HEK293T cells with CFIm59 or -68 KO kindly shared by Dr. Alan Engelman and Dr. Yonsheng Shi labs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Wisent). All cell lines were grown in a humidified incubator at 37°C with 5% CO₂. Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748). Transient transfection rescue of CFIm59 and CFIm68 in its respective KO cell line was performed using Lipofectamine 2000 (ThermoFisher) following the manufacturer's instruction and collected 24 h post-transfection.

Tseng H.W., Mota-Sydor A., Leventis R., Jovanovic P., Topisirovic I, & Duchaine T.F. (2022). Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade. Nucleic Acids Research, 50(16), 9397-9412.

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Adipogenesis under Hypoxia and Oxidized Water

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3T3-L1 pre-adipocytes were maintained in DMEM containing 10% calf serum (Hyclone Laboratories). Two-day post-confluent 3T3-L1 cells (designated Day 0) were induced to differentiate by the addition of a standard cocktail (MDI) composed of 0.5 mM IBMX, 1 μM Dex, and 10 μg/mL of insulin in 10% fetal bovine serum for 2 days. The cells were then cultured in DMEM supplemented with 10% FBS and 5 μg/mL of insulin for the next 6 days to allow the cells to become mature adipocytes. The medium was replaced by fresh medium every two days. For investigating the effect of OW on adipogenesis, culture media were prepared using freshly-prepared OW. Oil-Red O staining was performed as previously described [54 (link),55 (link),56 (link)]. For quantification, the dye was eluted by adding 100% isopropanol, and the extracts were analyzed by measuring the absorbance at 490 nm. For culturing the cells under a hypoxic environment, mature adipocytes were first cultured in serum-free media for 16 h then switched to the culture media prepared with either OW or regular water (RW) in a hypoxic chamber containing 1% O₂/94% N₂/5% CO₂ at 37 °C. The cells were harvested at the indicated time.

Cheng Y.J., Liu C.C., Chu F.Y., Yang C.P., Hsiao C.W., Chuang C.W., Shiau M.Y., Lee H.T., Tsai J.N, & Chang Y.H. (2020). Oxygenated Water Inhibits Adipogenesis and Attenuates Hepatic Steatosis in High-Fat Diet-Induced Obese Mice. International Journal of Molecular Sciences, 21(15), 5493.

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Cultivation of CHL Fibroblast Cell Line

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Chinese hamster lung fibroblast cell line (CHL) obtained from RIKEN BioResource Center (Ibaraki, Japan) was maintained in Eagle's medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 10% calf serum (HyClone Laboratories, Inc., South Logan, UT, USA), 2 mM L-glutamine (Wako Pure Chemical Industries, Ltd.), and 25 mM NaHCO 3 (Otsuka Pharmaceutical Factory, Inc.). Cells were cultured in a humidified atmosphere with 5% CO 2 at 37°C.

Kishino Y., Hasegawa T., Arakawa S., Shibaya Y., Yamoto T, & Mori K. (2019). Effect of the metabolic capacity in rat liver S9 on the positive results of in vitro micronucleus tests. The Journal of toxicological sciences, 44(3).

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3T3-L1 Fibroblast Differentiation Assay

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3T3-L1 fibroblasts were maintained in DMEM containing 10% calf serum (Hyclone Laboratories, South Logan, UT, USA) in an atmosphere of 5% CO₂ at 37 °C as previously described [7 (link),8 (link),12 (link),13 (link),14 (link),15 (link)]. Two days after fibroblasts had reached confluence, differentiation was induced by adding DMEM containing 0.5 mM IBMX, 10 μg/mL insulin and 1 μM Dex (MDI cocktail), and 10% fetal bovine serum (FBS) for 48 h. Cells were cultured in DMEM supplemented with 10% FBS and 5 μg/mL insulin for the next 6 days. Differentiation efficiency, cell morphology features and intracellular lipids were assayed by Oil Red O (ORO) staining on day 8. Briefly, cells were stained with ORO (0.6% dissolved in isopropanol and water, 6:4) for 30 min, then washed with distilled water. For quantification, lipid amounts were determined by measuring the absorbance of isopropanol-eluted dye at 490 nm.

Chang Y.H., Chang S.W., Hsu W.T., Yang C.P., Lo Y.L., Chen C.J., Tsai H.F, & Shiau M.Y. (2022). Implication of Adipogenesis-Coupled CRMP2 Functional Profile in Metabolic Homeostasis and Imbalance. Biomedicines, 10(10), 2603.

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Differentiation and Regulation of Mature Adipocytes

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3T3-L1 pre-adipocytes were allowed to differentiate into mature adipocytes as described^{17 (link),18 (link)}. In brief, the preadipocytes were cultured in DMEM containing 10% calf serum (Hyclone Laboratories, South Logan, Utah, USA). 2-day postconfluent (designated day 0), cells were induced to differentiate by adding 0.5 mM IBMX, 1 μM Dex, and 10 μg/ml insulin in 10% FBS for 2 days. The cells were then cultured in DMEM supplemented with 10% FBS and 5 μg/ml insulin for the next 6 days to allow the cells become terminally differentiated mature adipocytes^{17 (link),18 (link)}. For IL-4 treatment, Hsl mRNA levels were examined after the mature adipocytes were exposed to 10 ng/mL IL-4 for different time intervals after serum starvation for 2 h. For H89 experiments, mature adipocytes were pre-incubated with 20 μM H89 for 1 h and then treated with IL-4. To assess protein stability, mature adipocytes were treated with 10 μg/mL cycloheximide (CHX; C7698 from Sigma) in the presence or absence of IL-4. At the given time points, cells lysates were harvested and analyzed by Western blotting. Protein degradation rates were quantified by densitometry using time point zero as 100%.

Shiau M.Y., Chuang P.H., Yang C.P., Hsiao C.W., Chang S.W., Chang K.Y., Liu T.M., Chen H.W., Chuang C.C., Yuan S.Y, & Chang Y.H. (2019). Mechanism of Interleukin-4 Reducing Lipid Deposit by Regulating Hormone-Sensitive Lipase. Scientific Reports, 9, 11974.

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Isolation and Culture of Rat Hepatocytes

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Rat hepatocytes were harvested from male Sprague-Dawley rats (Harlan-Sprague-Dawley, Madison, WI) using collagenase perfusion and differential centrifugation as previously described (6 (link),7 (link)). The hepatocyte population was > 98% pure and had viability of > 95% (6 (link),7 (link)). All experimental protocols were approved by the University of Louisville Animal Care and Use Committee and followed guidelines prescribed by the National Institutes of Healths Guidelines for the Care and Use of Laboratory Animals. Hepatocytes were plated into 12-well or 100 mm gelatin-coated dishes at 2×10⁵ cells/well or 5×10⁶ cells/plate respectively in Williams medium E containing L-arginine (0.5 mM), insulin (10⁻⁶ M), HEPES (15 mM), L-glutamine, penicillin, streptomycin, and 10% low endotoxin calf serum (HyClone Laboratories, Logan, VT) After 4 hours, the cells were washed with PBS to remove nonadherent cells, the media replaced with insulin-free media containing 5% calf serum, and hepatocytes cultured overnight at 37°C. After overnight incubation, the cells were washed again with PBS to remove dead and nonadherent cells and the experimental conditions were established. Cultures were performed in duplicate or triplicate and experiments were repeated to ensure reproducibility.

Zhang B., Crankshaw W., Nesemeier R., Patel J., Nweze I., Lakshmanan J, & Harbrecht B.G. (2014). Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte inducible nitric oxide synthase expression. The Journal of surgical research, 193(2), 795-801.

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Olmesartan Cellular Stress Assay

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Alfa‐minimal essential medium (α‐MEM), penicillin 100 units/mL, and streptomycin 100 μg/mL were purchased from Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) and calf serum (CS) were purchased from HyClone Laboratories (Logan, UT, USA). Olmesartan was kindly supplied by Daiichi‐Sankyo Pharmaceutical Co. Ltd (Tokyo, Japan). Hydralazine and angiotensin II were purchased from Sigma (St. Louis, MO, USA). The fluorescent probe 2′,7′‐dihydro‐fluorescein diacetate (Molecular Probes, Eugene, OR, USA) was purchased from Invitrogen (Shibaura, Tokyo, Japan).

Wakamatsu T., Iwasaki Y., Yamamoto S., Matsuo K., Goto S., Narita I., Kazama J.J., Tanaka K., Ito A., Ozasa R., Nakano T., Miyakoshi C., Onishi Y., Fukuma S., Fukuhara S., Yamato H., Fukagawa M, & Akizawa T. (2020). Type I Angiotensin II Receptor Blockade Reduces Uremia‐Induced Deterioration of Bone Material Properties. Journal of Bone and Mineral Research, 36(1), 67-79.

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Spleen Tissue Lymphocyte Isolation

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Sterile spleen tissues were recovered from each group of rats on day 45 and ground into a homogenate (in 1 mL PBS buffer) that was centrifuged at 1,000 × g for 20 min at RT. The lymphocytes were removed and cell viability was assessed with trypan blue solution (survival rate >97%; BiYunTian). Last the lymphocytes in suspension (1~10 × 10⁶ cells/mL) were grown in culture medium containing 10% calf serum (Hyclone Laboratories, USA) at 37℃.

Li M., Ouyang W., Wu X., Zheng Y., Wei Y, & An L. (2014). Kinetin inhibits apoptosis of aging spleen cells induced by D-galactose in rats. Journal of Veterinary Science, 15(3), 353-359.

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