Scc066

Manufactured by Merck Group

The SCC066 is a laboratory equipment product manufactured by Merck Group. It is designed for precise and consistent measurements in scientific research and analysis. The core function of the SCC066 is to provide accurate and reliable data collection capabilities for laboratory applications.

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Lab products found in correlation

Scme004, by Merck Group (2 mentions) Hepg2, by American Type Culture Collection (1 mentions) Ccf sttg1, by American Type Culture Collection (1 mentions) Ab13970, by Abcam (1 mentions) Rat anti cd31, by BD (1 mentions) Endogro mv complete media kit, by Merck Group (1 mentions)

3 protocols using scc066

Generating Infectious HEV Strains for Research

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A subclone of the human hepatoma Huh7 cell line, Huh7-S10-3, a gift from Suzanne U. Emerson, NIH, Bethesda, MD, was used for generating infectious HEV RNA for transfection in this study. Another human hepatoma cell line, HepG2, was purchased from ATCC and used to propagate HEV. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. A human astrocyte-derived cell line (CCF-STTG1) was purchased from ATCC and cultured in DMEM with 10% FBS. A human brain microvascular endothelial cell line (hCMEC/D3), which is derived from human temporal lobe microvessel endothelial cells (Millipore, SCC066), was cultured in a collagen-coated flask or plate with hCMEC/D3-specific EndoGRO-MV complete media (Millipore, SCME004) containing human fibroblast growth factor (65 (link)). Genotype 3 HEV strain Kernow-C1/P6 (designated as P6) was rescued by transfection of Huh7-S10-3 cells with in vitro-transcribed RNAs from an infectious complementary DNA (cDNA) clone of the HEV P6 strain (66 (link)). The virus stock of another human-origin genotype 3 HEV (strain US2) was prepared in 10% PBS suspension of feces from experimentally infected pigs (67 (link)).

Tian D., Li W., Heffron C.L., Wang B., Mahsoub H.M., Sooryanarain H., Hassebroek A.M., Clark-Deener S., LeRoith T, & Meng X.J. (2022). Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood–brain barrier, and invades the central nervous system. Proceedings of the National Academy of Sciences of the United States of America, 119(24), e2201862119.

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Infection of Brain Endothelial Cells

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Primary brain endothelial cells of mice (8 weeks of age) were cultured in 24‐well plates as described previously (Ridder et al, 2011). Freshly purchased immortalized human brain endothelial cells (hCMEC/D3, free of mycoplasma, hepatitis A, B, C, HPV, herpes, and HIV 1 and 2; Millipore #SCC066) were grown in 24‐well plates coated with rat tail collagen, type I (1:20 in 1× PBS) in EndoGRO‐MV medium (Millipore # SCME004) supplemented with 1 ng/ml FGF2 (Millipore #GF003). Primary murine cells were infected with 1.0 × 10¹⁰ gp/well, three days after preparation. Immortalized human cells were infected with 1.6 × 10¹⁰ gp/well, one day after seeding. Medium was changed at least 3 days after infection. Ten days (murine cells) or 4 days (human cells) after infection, cells were fixed in 4% PFA and immunostained with chicken anti‐GFP (1:2,000, Abcam #ab13970), and DAPI (1:2,000). Murine cells were additionally stained with rat anti‐CD31, 1:500 (BD Pharmingen #557355). Infectivity was determined by the ratio of GFP‐positive cells to DAPI.

Körbelin J., Dogbevia G., Michelfelder S., Ridder D.A., Hunger A., Wenzel J., Seismann H., Lampe M., Bannach J., Pasparakis M., Kleinschmidt J.A., Schwaninger M, & Trepel M. (2016). A brain microvasculature endothelial cell‐specific viral vector with the potential to treat neurovascular and neurological diseases. EMBO Molecular Medicine, 8(6), 609-625.

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Synchronized Brain Endothelial Cell Culture

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Immortalized human brain capillary endothelial cells (Millipore SCC066) were cultured in EndoGRO-MV Complete media kit (SCME004) as recommended by the manufacturer. Cells were tested every 3–5 passages for mycoplasma contamination by DNA Hoechst stain. Cultureware was coated with rat tail collagen type I solution at a concentration of 0.1 mg/mL and was incubated for >1 h at 25 °C. Cells were cultured in an incubator at 37 °C with 5% CO₂. For synchronization of culture, cells were grown to ~60% confluence and incubated in 200 nM dexamethasone for 30 min. Circadian time points were obtained by staggering the synchronization of different cultures and assaying them at the same time.

Zhang S.L., Lahens N.F., Yue Z., Arnold D.M., Pakstis P.P., Schwarz J.E, & Sehgal A. (2021). A circadian clock regulates efflux by the blood-brain barrier in mice and human cells. Nature Communications, 12, 617.

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