Human B-cell [RPMI8866 and Tat-GFP expressing RPMI8866 (RPMI8866-Tat-GFP)] and T-cell (Jurkat) lines were used in the study. Cells were maintained in EX-CELL Medium (SAFC Biosciences, United States) supplemented with L-glutamine and 10% FBS (Paneco, Russia). The effects of three MT inhibitors on cell cycle and cell death were evaluated in the study: paclitaxel (taxol; Sigma, United States), nocodazole (Biochem, United States) and vinorelbine (Sigma, United States). MT inhibitors were added to the medium 24 h prior to analysis at final concentrations of 3; 10; 30; 100; 300; and 1000 nM.
Fetal bovine serum (fbs)
Manufactured by PanEco
Sourced in United States
Fetal bovine serum is a widely used cell culture supplement. It is derived from the blood of bovine fetuses and provides a rich source of proteins, growth factors, and other nutrients essential for cell growth and proliferation in vitro.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by PanEco (10 mentions)
Penicillin, by PanEco (8 mentions)
Streptomycin, by PanEco (8 mentions)
Dulbecco modified eagle medium (dmem), by PanEco (8 mentions)
Trypsin, by PanEco (6 mentions)
Penicillin streptomycin, by PanEco (6 mentions)
Amphotericin b, by PanEco (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Sodium pyruvate, by PanEco (3 mentions)
Ethylene diamine tetraacetic acid (edta), by PanEco (3 mentions)
D glucose, by Merck Group (3 mentions)
Trypsin edta, by PanEco (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by PanEco (2 mentions)
Chaps, by Merck Group (2 mentions)
Scp0139, by Merck Group (2 mentions)
Ac devd afc, by Bio-Techne (2 mentions)
Ac lehd afc, by Bio-Techne (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
22 protocols using fetal bovine serum (fbs)
1
Microtubule Inhibitor Effects on Cell Lines
2
Metastatic Breast Cancer Cell Lines
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We have used two commercially available human epithelial cell lines from breast cancer with high (MDA-MB-231) and low (MCF7) metastatic potential (from American Tissue Culture Collection (ATCC), Manassas, VA). Cell lines were cultured in their appropriate media, as recommended by ATCC: we used Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, Gibco, Waltham, MA) supplemented with 10 vol% fetal bovine serum (FBS) (Dia-M, Moscow, Russia), and 1 vol% each of L-glutamine (OOO NPP PanEco, Moscow, Russia), penicillin–streptomycin (OOO NPP PanEco, Moscow, Russia), and sodium pyruvate (OOO NPP PanEco, Moscow, Russia). Benign cells (MCF10-A), used as control, were grown in DMEM/Ham’sF-12 medium supplemented with 5% horse serum (Hyclone, Waltham, MA),1vol.% of l-glutamine and penicillin/streptomycin, 10 μg/ml insulin, 10 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, and 100 ng/ml cholera toxin; experiments were run with FBS containing media. Cells were cultured at 37 C, 5% CO2, and high humidity. No mycoplasma testing was done. Cells were frozen at low passages from ATCC stock (i.e., 3–5), and for experiments, cells were thawed and used in passages 7–30 from the ATCC stock.
3
SARS-CoV-2 Variant Antibody Phagocytosis Analysis
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An assessment of antibody-dependent monocyte-mediated phagocytosis (ADMP) was performed on the THP-1 cells. For this, the recombinant S-proteins of D614G, B.1.617.2, and BA.1 SARS-CoV-2 variants (Miltenyi Biotec) were biotinylated using a protein-biotinylating kit (Lumiprobe), followed by incubation for 12 h with fluorescent 1 µm neutravidin particles (Invitrogen, F8775) at 4 °C. Next, the resultant particles were incubated with heat-inactivated serum samples at 37 °C for 2 h. The THP-1 cells were maintained in RPMI medium (PanEco) supplemented with 10 mM L-Glutamine and 10% FBS (PanEco). A total of 25 × 104 of the THP-1 cells were seeded in a 96-well plate in serum-free AIM-V medium (Gibco), and then the S-protein-covered fluorescent particles were added and incubated overnight at 37 °C, 5% CO2. Next, the cells were washed with PBS, fixated with 4% PFA, and analyzed using flow cytometry using the CytoFlex LX (Beckman Coulter). The antibody-dependent phagocytic activity index was calculated as follows: (% of cells containing particles × MFI (mean fluorescent intensity) of cells containing particles)/104 [24 (link),25 (link)].
4
Generation of SARS-CoV-2 Pseudotyped Lentivirus
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Pseudotyped lentiviral particles were generated by the co-transfection of HEK293T cells with psPAX2 (addgene #12260), pLV-PGK-GFP plasmids, and a plasmid carrying the corresponding S-protein sequence. 24 h after -transfection, the cultural media was changed to virus-producing medium (Opti-MEM (Gibco), 5% FBS (PanEco), and 1 mM sodium pyruvate (PanEco)). Next, after 24 h, the virus-containing supernatant was collected, filtered through a 0.45 μm polyester-sulfone filter, frozen, and stored at −80 °C. The resultant pseudotyped particles were titrated on HEK293T-hACE2-TMPRSS2 cells, and the infection percentage was determined by flow cytometry. The titers of the pseudotypes were equalized before the neutralization assay.
5
Inflammatory Signaling Pathway Analysis
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LPS (Sigma-Aldrich, St. Louis, MO, USA), rosiglitazone (Sigma-Aldrich), streptomycin–penicillin, trypsin, EDTA, fetal bovine serum and Culture medium Dulbecco’s Modified Eagle Medium (DMEM) were from PanEco (Moscow, Russia). Antibodies against COX-2 (Cell Signaling Technology, D5H5, Danvers, MA, USA), phospho-p38 (Cell Signaling Technology, 4511), p38 (Cell Signaling Technology, 9212), p-JNK (cat.no sc-12882), JNK (cat.no sc-571), and β-actin (cat.no sc-47778) (Santa Cruz Biotechnology, CA, USA (SCBT)); secondary horseradish peroxidase conjugated antibodies (anti-rabbit, anti-mouse, and anti-goat) (SCBT and CST); Western Blotting Substrate ECL (Thermo Fisher Scientific, MA, USA); and ELISA kits for PGE2, TNFα and IL-10 (Thermo Fisher Scientific) were also used.
6
Cell Culture Conditions and Maintenance
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Cell lines were obtained from Blokhin CRC cell collection. Cells were cultured in DMEM (for adhesion cell cultures) or RPMI-1640 (for suspension cell cultures) supplemented with L-glutamine (0.584 mg/mL), penicillin (50 U/mL) and streptomycin (50 μg/mL) (PanEco, Moscow, Russia) and 10% fetal bovine serum (PanEco, Russia). Cell lines were incubated at 37 °C in 5% CO2.
7
Culturing Human Cell Lines
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Human cervical cancer cells (HeLa), fibrosarcoma cell line (HT1080), immortalized human keratinocyte cells (HaCaT), and immortalized kidney epithelial cell line (NKE-hTERT) were obtained from the Blokhin CRC cell collection. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine (0.584 mg/mL), penicillin (50 U/mL), and streptomycin (50 μg/mL) (PanEco, Moscow, Russia) and 10% fetal bovine serum (PanEco). Cell lines were incubated at 37 °C and 5% CO2.
8
Evaluating PBMC Viability in Culture
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The viability of human peripheral blood mononuclear cells was studied.
At first, 10 mL of blood was diluted 1:1 with PBS, layered on ficoll and centrifuged at 2000 rpm for 30 min. Thus, a layer containing mononuclear cells was obtained. Then, 5 mL of the buffy coat sample were washed twice with 10 mL of PBS, centrifuged at 1500 rpm for 5 min, and placed in a 12-well plate containing 2 × 106 cells/mL of RPMI-1640 (Paneco, Moscow, Russia). We added to this 10% fetal bovine serum (Paneco, Moscow, Russia), antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) and 2 mM L-glutamine at 37 °C in a 5% CO2 atmosphere. The studied samples were placed in a 12-well plate and incubated for 24 and 72 h. The viability of this product was then determined using the trypan blue dye exclusion method.
At first, 10 mL of blood was diluted 1:1 with PBS, layered on ficoll and centrifuged at 2000 rpm for 30 min. Thus, a layer containing mononuclear cells was obtained. Then, 5 mL of the buffy coat sample were washed twice with 10 mL of PBS, centrifuged at 1500 rpm for 5 min, and placed in a 12-well plate containing 2 × 106 cells/mL of RPMI-1640 (Paneco, Moscow, Russia). We added to this 10% fetal bovine serum (Paneco, Moscow, Russia), antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) and 2 mM L-glutamine at 37 °C in a 5% CO2 atmosphere. The studied samples were placed in a 12-well plate and incubated for 24 and 72 h. The viability of this product was then determined using the trypan blue dye exclusion method.
9
Transient Transfection of HeLa Cells
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HeLa Kyoto cells (ATCCs) were grown in Dulbecco’s modified medium (DMEM; PanEco, Moscow, Russia) with 10% (v/v) fetal bovine serum (PanEco) containing 50 U/mL penicillin and 50 μg/mL streptomycin (PanEco) at 37 °C and 5% CO2 and seeded onto a 35 mm glass-bottomed culture dish (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Republic of Korea) 24 h before transfection. Polyethylenimine (PEI, #23966-1, Polysciences, Warrington, PA, USA) was used as a transfection reagent. For transfection, DMEM was changed for Opti-MEM one hour before the procedure. In total, 3 μL of PEI was mixed with 250 μL of Opti-MEM per dish, and in a separate tube 1 μg of plasmid DNA was mixed with 250 μL of Oti-MEM. Mixtures were incubated for 5 min, and then PEI- and DNA-containing media were mixed and the resulting solution was incubated for 20 min. These procedures were carried out at room temperature. PEI-DNA mixture was added to cells dropwise, cells were incubated for 3 h at 37 °C and 5% CO2, and transfection media were replaced with DMEM complete.
10
Cytotoxicity Evaluation of Therapeutic Compounds
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L-glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, Hanks’ salts, Earle’s salts, trypsin, DMEM, MEM, and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), isopropanol, HCl, CHAPS, protease inhibitor cocktail, EDTA, dithiothreitol, HEPES, DMSO, SCP0139, toluene, acetonitrile, carbon tetrachloride, diethylenetriamine, urea, triethylamine, 4-chlorophenyl isocyanate, 3,4-dichlorophenyl isocyanate, and D-glucose were from Sigma-Aldrich, St. Louis, MO, USA. Hydroxychloroquine, Z-VAD-FMK, necrostatin-1, necrosulfonate, NQDI-1, Ac-DEVD-AFC, and Ac-LEHD-AFC were from Tocris Bioscience, Bristol, UK. The apoptosis assay kit was from Abcam, Cambridge, MA, USA.
Cell lines were purchased from ATCC, Manassas, VA, USA.
Cell lines were purchased from ATCC, Manassas, VA, USA.
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