Sim hf medium

Manufactured by Sino Biological

Sourced in China

SIM HF medium is a cell culture medium formulated to support the growth and maintenance of hybridoma cells. It is a highly concentrated, serum-free, and protein-free medium designed to provide the necessary nutrients and growth factors for efficient hybridoma cell culture.

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Lab products found in correlation

6 protocols using sim hf medium

Recombinant Expression and Purification of Drosophila Apoptotic Proteins

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The full-length D. melanogaster Dark complementary DNA was subcloned into vector pFastBac1 (Invitrogen) with a C-terminal 10xHis tag. The recombinant Dark was expressed using the Bac-to-Bac baculovirus system (Invitrogen). Bacmid DNAs were generated in DH10Bac cells, and the resulting baculoviruses were generated and amplified in Sf9 insect cells (Invitrogen). Dark protein was overexpressed in Hi-5 insect cells (Invitrogen) grown in the SIM HF medium (Sino Biological, Inc.). Forty-eight hours after viral infection, the cells were collected and homogenized in a buffer containing 25 mM Tris (pH 8.0) and 150 mM NaCl. The cells were disrupted by 60 strokes on ice using a Dounce homogenizer. After high-speed centrifugation at 14,000 rpm for 60 min, the supernatant was harvested. The Dark protein was purified to homogeneity by nickel affinity chromatography (Ni-NTA, Qiagen) and anion exchange chromatography (Source-15Q, GE Healthcare).
The full-length Dronc (residues 1–450), Dronc-CARD (residues 1–130), and relevant mutants were subcloned into pET-21b with a C-terminal 8xHis tag. All mutants were generated using a standard PCR-based strategy and verified by plasmid sequencing. All Dronc proteins and mutants were expressed in Escherichia coli strain BL21(DE3) and purified to homogeneity as described (Yan et al. 2006 (link)).

Pang Y., Bai X.C., Yan C., Hao Q., Chen Z., Wang J.W., Scheres S.H, & Shi Y. (2015). Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila. Genes & Development, 29(3), 277-287.

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Cell Culture Maintenance Protocol

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Sf21 and High Five cells, originally purchased from Invitrogen, were maintained using a non-humidified shaker at 27 °C in the SIM SF medium and the SIM HF medium (Sino Biological Inc.), respectively. U2OS cells, originally purchased from ATCC, were grown in a humidified incubator with 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) with 100 μg per ml penicillin/streptomycin (GIBCO). All cell lines were tested to be free of mycoplasma by the standard PCR method. Identity of the cell lines were frequently assessed by their morphological features.

Zhang H., Zhu Q., Cui J., Wang Y., Chen M.J., Guo X., Tagliabracci V.S., Dixon J.E, & Xiao J. (2018). Structure and evolution of the Fam20 kinases. Nature Communications, 9, 1218.

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Isolation and Propagation of PDCoV

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Hi5 and sf9 insect cells were maintained in the SIM HF medium and SIM SF medium (Sino Biological Inc., Beijing, China) at 27 °C, respectively. LLC-PK1 cells and BHK-21 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% and 10% fetal bovine serum (Tianhang Biotech, Hangzhou, China), respectively, at 37 °C with 5% CO₂.
PDCoV CZ2020 strain (GenBank accession No. OK546242) was isolated from a piglet suffering severe diarrhea and passaged in LLC-PK1 cells with DMEM supplemented with 7.5 μg/ml trypsin. The porcine anti-PDCoV hyperimmune serum was produced in the lab.

Ji W., Peng Q., Fang X., Li Z., Li Y., Xu C., Zhao S., Li J., Chen R., Mo G., Wei Z., Xu Y., Li B, & Zhang S. (2022). Structures of a deltacoronavirus spike protein bound to porcine and human receptors. Nature Communications, 13, 1467.

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Cultivation and Purification of SVCV

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Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) at 37°C in 5% CO₂. Epithelioma papulosum cyprini (EPC) cells were cultured in Medium 199 (M199, Gibco) supplemented with 10% (v/v) FBS at 28°C in 5% CO₂. Spodoptera frugiperda (Sf9) cells were cultured in SIM HF medium (Sino Biological) supplemented with 10% (v/v) FBS at 28°C without CO₂. Spring viremia of carp virus (SVCV) was a gift from Prof. Yibing Zhang (Institute of Hydrobiology, Chinese Academy of Sciences). SVCV was propagated in EPC cells at 28°C and titrated in 96-well plates until the cytopathic effect (CPE) was complete. SVCV was purified from EPC culture medium by sucrose gradient centrifugation at 100,000 g for 2 h as previously described [65 (link)], and finally suspended in sterile PBS and stored at -80°C until use. Viral titer was determined to be 1.58 × 10⁷ TCID₅₀/mL by 50% tissue culture infective dose (TCID₅₀) assay on EPC cells using the Reed-Müench method [66 ]. For vaccination purpose, SVCV was inactivated by formaldehyde with a final concentration of 0.4% (v/v) followed by the protocol as described [67 (link)].

Hong Y., Hu C.B., Bai J., Fan D.D., Lin A.F., Xiang L.X, & Shao J.Z. (2023). Essential role of an ERV-derived Env38 protein in adaptive humoral immunity against an exogenous SVCV infection in a zebrafish model. PLOS Pathogens, 19(4), e1011222.

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Production and Purification of Recombinant Human Fam20A

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DNA fragment encoding Human Fam20A residues 69–529 was cloned into a modified pI-secSUMOstar vector (LifeSensors, Malvern, PA) containing a tobacco etch virus (TEV) protease cleavage site. Bacmids were generated using the Bac-to-Bac system (Invitrogen, Carlsbad, CA). Recombinant baculoviruses were generated and amplified using the Sf21 insect cells (RRID: CVCL_0518), maintained in the SIM SF medium (Sino Biological Inc., Beijing, China). For protein production, Hi5 cells (RRID: CVCL_C190) grown in the SIM HF medium (Sino Biological Inc.) were infected at a density of 1.5–2.0 × 10⁶ cells/ml. 48 hr post infection, 2 liters of conditioned medium were collected by centrifugation at 200x g. The medium was concentrated using a Hydrosart Ultrafilter (Sartorius, Göttingen, Germany) and exchanged into the binding buffer containing 25 mM Tris-HCl, pH 8.0, 200 mM NaCl. The proteins were then purified using the Ni-NTA resin (GE healthcare, Chicago, IL). The N-terminal 6xHis-SUMO fusion tag was removed by the TEV protease. Untagged Fam20A was further purified by the anion exchange chromatography using a Resource Q column (GE healthcare), followed by the size-exclusion chromatography using a Superdex 200 increase 10/300 GL column (GE healthcare).

Cui J., Zhu Q., Zhang H., Cianfrocco M.A., Leschziner A.E., Dixon J.E, & Xiao J. (2017). Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding. eLife, 6, e23990.

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Cell Culture Protocols for Various Organisms

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All Escherichia coli strains were cultured in lysogeny broth medium (1% w/v tryptone, 0.5% w/v yeast extract and 1% w/v NaCl) using a non-humidified shaker at 37 °C. Spodoptera frugiperda (Sf9) cells and Trichoplusia ni (Hi5) cells were maintained in SIM SF medium and the SIM HF medium (Sino Biological), respectively, using a non-humidified shaker at 27 °C. HEK293T and Huh-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin and 0.1 mg/ml of streptomycin with 5% CO 2 at 37 °C.

Yang J., Wang W., Chen Z., Lu S., Yang F., Bi Z., Bao L., Mo F., Li X., Huang Y., Hong W., Yang Y., Zhao Y., Ye F., Lin S., Deng W., Chen H., Lei H., Zhang Z., Luo M., Gao H., Zheng Y., Gong Y., Jiang X., Xu Y., Lv Q., Li D., Wang M., Li F., Wang S., Wang G., Yu P., Qu Y., Yang L., Deng H., Tong A., Li J., Wang Z., Yang J., Shen G., Zhao Z., Li Y., Luo J., Liu H., Yu W., Yang M., Xu J., Wang J., Li H., Wang H., Kuang D., Lin P., Hu Z., Guo W., Cheng W., He Y., Song X., Chen C., Xue Z., Yao S., Chen L., Ma X., Chen S., Gou M., Huang W., Wang Y., Fan C., Tian Z., Shi M., Wang F.S., Dai L., Wu M., Li G., Wang G., Peng Y., Qian Z., Huang C., Lau J.Y., Yang Z., Wei Y., Cen X., Peng X., Qin C., Zhang K., Lu G, & Wei X. (2020). A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature, 586(7830).

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