Glioma cell lines (U251, LN229, U87, and A172) and HUVECs were obtained from the Neurosurgery Laboratory of the First Affiliated Hospital of Harbin Medical University. Glioma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; D6429, Sigma, USA) with 10% fetal bovine serum (FBS; 16000-044, Gibco, USA), and Roswell Park Memorial Institute (RPMI)-1640 medium (RPMI-1640; R8758, Sigma, USA) containing 10% FBS was utilized to culture the HUVEC cell line. Finally, these cell lines were placed in a 37 °C incubator with 5% CO2.
R8758
Manufactured by Merck Group
Sourced in United States
R8758 is a laboratory equipment product manufactured by Merck Group. It serves as a core component for various scientific applications. The detailed technical specifications and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
P4333, by Merck Group (5 mentions)
D6429, by Merck Group (3 mentions)
F7524, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
S8636, by Merck Group (2 mentions)
M7145, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
A2942, by Merck Group (2 mentions)
S0615, by Merck Group (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Emem f 12, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
E2888, by Merck Group (1 mentions)
M4655, by Merck Group (1 mentions)
Countess 2, by Thermo Fisher Scientific (1 mentions)
T8154, by Merck Group (1 mentions)
F0926, by Merck Group (1 mentions)
G7513, by Merck Group (1 mentions)
34 protocols using r8758
1
Glioma and HUVEC Cell Culture
2
Culturing Human Neuroblastoma Cell Lines
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IMR-32 human neuroblastoma cell line was cultured in EMEM medium (M4655, Sigma-Aldrich) supplemented with 10% fetal calf serum (10270106, Gibco), 1% non-essential amino acid solution (M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 50 µg/ml gentamicin (G1397, Sigma-Aldrich). CHP-134 cells were grown in RPMI 1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FCS and 50 µg/ml gentamicin. LAN-1 cells were cultured in EMEM/F-12 (N6658, Sigma-Aldrich) medium diluted in 1:1 ratio, supplemented with 10% FCS, 1% non-essential amino acid solution, 1 mM sodium pyruvate and 50 µg/ml gentamicin, while LAN-5 cells in RPMI 1640 medium supplemented with 20% FCS and 50 µg/ml gentamicin. For preparation of positive controls, IMR-32 and CHP-134 cells were cultured in amino acids-deprived Earle’s Balanced Salt (E2888, Sigma-Aldrich), supplemented with 10% FCS for 24 h. All cell lines were grown at 37 °C in a 5% CO2 atmosphere.
3
Mesothelioma Cell Culture Protocol
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Five mesothelioma cell lines and an immortalized human mesothelial cell line (Table 1 ) were cultured in growth medium consisting of Roswell Park Memorial Institute (RPMI) 1640 with L-glutamine and sodium bicarbonate (R8758; Sigma–Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (F9423; Sigma–Aldrich, MO, USA). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2. Cell viability was assessed using trypan blue exclusion methods (T8154; Sigma–Aldrich, MO, USA). Cell viability and cell counts were obtained using an automated cell counter (Countess II, Thermo Fisher Scientific, Waltham, MA, USA).
4
Zika-specific T cell immune response in mice
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At 18 days post-primary infection, spleens from ZIKV infected HLA-A2 male and female mice were ground over a 70μm cell strainer and washed with RPMI (Sigma Aldrich R8758) supplemented with 10% fetal bovine serum (Sigma Aldrich F0926), 1% HEPES (Sigma Aldrich H3537), and 1X beta-mercaptoethanol (Gibco 21985023). Approximately 106 cells were stimulated in the presence of 10 μg per ml of brefeldin A for 6 hours in each well of a 96 well round bottom plate with 10 μg of pooled 9-mer or 15-mer peptides (21st Century Biochemicals). The peptides used were previously published as ZIKV MHC class I and II H2b restricted epitopes respectively [35 (link),36 (link)].
5
Culturing A549 Lung Cancer Cells
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Commercially accessible epithelial lung carcinoma cells from the ATCC were used in this investigation, A549 (ATCC®, CCL185TM). The media used for maintaining the lung cancer cells and isolated CSCs comprised the accompanying: Rosewell Park Memorial Institute 1640 medium (RPMI-1640) (SIGMA, R8758) enhanced with 10% fetal bovine serum (Biochrom, S0615) and 0.5% penicillin/streptomycin (SIGMA, P4333) and 0.5% amphotericin B (SIGMA, A2942). All cultured cells were maintained and incubated at 37°C in 5% CO2 and 85% humidity.
6
Culturing Metastatic Melanoma and HEK 293 Cells
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WM-266-4 human metastatic melanoma cell line and HEK 293 cell line were cultured in RPMI medium (R8758, Sigma, Rehovot, Israel) supplemented with 2 mM glutamine (G7513, Sigma, Rehovt, Israel), 10% fetal bovine serum (F7524, Sigma, Rehovot, Israel) and penicillin and streptomycin (100 IU/ml each) (P4333, Sigma, Rehovot, Israel).
7
Hoechst Staining for Nuclear Morphology
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Any alterations in the nuclear morphology of the treated and untreated cells were assessed by the Hoechst stain assay. Cells were seeded in 3.4 cm diameter culture dishes over sterile coverslips and allowed to reach above 80% confluence. Once confluent, cells were exposed to the different treatments. After 24 h of incubation at 37°C with 5% CO2 and 85% humidity, each coverslip was stained with 200 μl of the Hoechst working stock solution (Hoechst 33258, Invitrogen, H1398) and incubated at room temperature for 30 min. The solution was removed from each coverslip, and cells were rinsed with phosphate-buffered saline (PBS, Sigma-Aldrich, R8758) to remove any remaining stain. Fluorescence was observed in a dark room with a fluorescent microscope (Axio observer Z1, Carl Zeiss) using a broadband 4′,6-Diamidino-2-Phenylindole (DAPI) filter set, measured at an excitation wavelength of 352 nm and an emission wavelength of 455 nm.
8
Isolation and Culture of Murine Macrophages and Neutrophils
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Mice were euthanized by CO2 inhalation and dissected femurs and tibias. Bone marrow was collected by PBS flushing. Cells were centrifuged at 1000rpm for 5 min. Then, supernatant was removed. Cells were resuspended in red blood cell lysis buffer (5 mL, Beyotime #C3702) for 5 min at room temperature. Added PBS (5mL) and centrifuged at 1000rpm for 5 min. Cells were resuspended and cultured in low glucose DMEM (sigma #D6046) with macrophage colony stimulating factor (MCSF) (25 ng/mL, Abcam #ab129146) and 10% FBS. After 3 days of cell culture, the fresh culture medium was replaced, and then cultured for 4 days, bone marrow-derived macrophage (BMDM) could be used for subsequent experiments.
For neutrophil culture, peripheral blood of mice was collected and centrifuged at 500g for 5 min to separate serum. Added the same volume of 0.9% NaCl solution and mixed. Ficoll neutrophil isolation solution (TBD #LST1077-1) was used according to the manufacturer’s instructions. Next, layer of red blood cell and neutrophil was collected. Cells were centrifuged at 1200rpm for 5 min and resuspended in red blood cell lysis buffer (Beyotime #C3702) for 5 min at room temperature (the process was repeated three times). Then, cells were cultured in 1640 medium (sigma #R8758) with 10% FBS and serum.
For neutrophil culture, peripheral blood of mice was collected and centrifuged at 500g for 5 min to separate serum. Added the same volume of 0.9% NaCl solution and mixed. Ficoll neutrophil isolation solution (TBD #LST1077-1) was used according to the manufacturer’s instructions. Next, layer of red blood cell and neutrophil was collected. Cells were centrifuged at 1200rpm for 5 min and resuspended in red blood cell lysis buffer (Beyotime #C3702) for 5 min at room temperature (the process was repeated three times). Then, cells were cultured in 1640 medium (sigma #R8758) with 10% FBS and serum.
9
Rat pheochromocytoma (PC12) cell culture and treatment
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The adherent sub-clone of rat pheochromocytoma (PC12) cells were maintained as described previously [51 (link)] in RPMI 1640 medium (R-8758, Sigma-Aldrich, Saint-Louis, USA), with 10% (v/v) horse serum (PAA Laboratories GmbH, GE Healthcare, Chicago, USA), 5% (v/v) foetal bovine serum (F-9665, Sigma-Aldrich, Saint-Louis, USA), 100 units/ml penicillin, and 100 μg/mL streptomycin (P-0781; Sigma-Aldrich, Saint-Louis, USA) in a humidified 5% CO2 atmosphere at 37°C . The cells were treated with 100 μM EGTA (E-4378; Sigma-Aldrich, Saint-Louis, USA) and/or 20 μM forskolin (344282; Sigma-Aldrich, Saint-Louis, USA) for 1 h or 2 h, as indicated. Cells were briefly washed with PBS and lysed in Lysis buffer (50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v) NP-40, 1 mM dithiothreitol, 1 mM Na3VO4, 1 mM NaF, and 1x EDTA-free protease inhibitor cocktail from Roche (11836170001; Roche, Basel, Switzerland).
10
Cell Culture and Transfection Protocol
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