HeLa cells were provided from the University of South of China and cultured in complete DMEM (Gibico; Thermo Fisher Scientific, Inc), containing 10% FBS (Gibico; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO2. HeLa cells were infected with C. trachomatis. C. trachomatis was added into HeLa cell. At 48 h post-infection, the cells were washed twice with cold PBS, and lysed with RIPA buffer (Epizyme, Inc.) for 15 min at 4°C. The lysate was pre-clarified by rotation for 10 min, centrifuged at 14,000 × g for 15 min (both 4°C), and incubated with protein-A agarose beads. Cell-free lysates were incubated with a rabbit anti-PHB antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. ab75766.) or IgM antibody (1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Protein-A beads were added for 2 h at room temperature, and the lysates were subsequently washed three timeswith PBS. The proteins were eluted into 5X SDS-PAGE sample buffer, separated by 12% SDS-PAGE gel for immunoblotting.
Gibico
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gibco is a line of cell culture media, supplements, and reagents produced by Thermo Fisher Scientific. Gibco products are designed to support the growth and maintenance of a wide variety of cell types in cell culture applications. The core function of Gibco products is to provide the necessary nutrients, growth factors, and other components required for optimal cell growth and development in a controlled laboratory setting.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
Multi detection microplate reader, by Agilent Technologies (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ripa buffer, by Ipsen (1 mentions)
Human pdgf bb, by R&D Systems (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Nci h1299, by American Type Culture Collection (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
White 96 well plate, by Corning (1 mentions)
Weri rb 1, by National Collection of Authenticated Cell Cultures (1 mentions)
10 protocols using gibico
1
Chlamydia trachomatis Infection in HeLa Cells
2
Splenocyte Viability Assay for BTWT Components
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SplenocyteS isolated from the spleen of mice were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin, and streptomycin (Gibico, Invitrogen, Carlsbad, CA, USA) overnight. To evaluate the cell viability, 2 × 105 splenocytes cultured in 96-well plates were treated with 1 µM of 11 components of BTWT for 24 h and then submitted to cell viability assay by mitochondria-dependent reduction of thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich). All experiments were repeated in quadruplicate in different intervals.
3
Cytotoxicity Evaluation of PEG Compounds
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A cell counting kit-8 (CCK-8, Beyotime Institute of Biotechnology, China) was used to investigate the cytotoxicity of N3–PEG3–N3 and Tetra-PEG-DBCO against 4T1 cells. Briefly, cells were seeded at a density of 1 × 104 cells per well in a 96-well plate, and cultured overnight in RMPI 1640 (Hyclone, Thermo Scientific) supplemented with 10% fetal bovine serum (Hyclone, Thermo Scientific) and 1% penicillin & streptomycin (GIBICO, Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2. Subsequently, 100 μl of fresh medium containing N3–PEG3–N3 at different concentrations (100, 500, or 1000 μg/ml) or Tetra-PEG-DBCO at different concentrations (6000, 10000, or 60000 μg/ml) was added to replace the culture medium for 24 and 48 h, respectively. After incubation, the culture medium containing N3–PEG3–N3 or Tetra-PEG-DBCO was removed, 100 μl of fresh medium and 10 μl of CCK-8 solution were added to each well. After incubation for 3 h, the OD value of cultures was recorded at 450 nm with the help of a Multi-Detection Microplate reader (BioTek, USA). The well with medium and CCK-8 solution but without cells, N3–PEG3–N3, and Tetra-PEG-DBCO was used as a blank group. The well with medium, cells, and CCK-8 solution but without N3–PEG3–N3 and Tetra-PEG-DBCO was employed as a control group. The experiment was replicated for four parallel samples.
4
Osteogenic Differentiation of Human BMSCs
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Human BMSCs were isolated from healthy volunteers and expanded following the methods previously reported28 (link). The BMSCs prior to passage four were used in the following experiments. An osteogenic medium consists of α-MEM supplemented with 10% FBS, 50 mg/ml L-ascorbic acid, 10 mM glycerophosphate, and 100 nM dexamethasone and antibiotics (Sigma; St. Louis, MO, USA) is used for osteogenic differentiation induction. The maintaining medium consists of DMEM (Gibico, Invitrogen) with 10% FBS and 100 U/mL penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO2.
The expression of miRNA was achieved by transfecting BMSCs with miR-381-3p mimics or miR-381-3p inhibitor (Invitrogen) in six-well plates at a density of 4 × 105/well with the help of Lipofectamine RNAiMAX (Invitrogen).
The expression of miRNA was achieved by transfecting BMSCs with miR-381-3p mimics or miR-381-3p inhibitor (Invitrogen) in six-well plates at a density of 4 × 105/well with the help of Lipofectamine RNAiMAX (Invitrogen).
5
Culturing Human Breast Cancer MCF-7 Cells
6
Isolation and Culture of Smooth Muscle Cells
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As previously described, HSVSMCs were isolated from saphenous vein segments obtained from patients undergoing CABG surgeries [23 (link), 24 (link)]. For cell culture, SMC medium (Siencell, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum was used (FBS; Gibico, Thermo Fisher Scientific, Waltham, MA, USA). HSVSMCs was verified by immunofluorescence staining for smooth muscle-specific α-actin (SMα-actin) and smooth muscle 22α (SM22α). All experiments employed HSVSMCs at passages 3–5. For PDGF-BB treatment, HSVSMCs were cultured to 70–80% confluence and then serum-starved for 24 h before being incubated with human PDGF-BB for 24 h again (R&D Systems, Minneapolis, MN, USA) in SMC medium containing 0.5% FBS, as previously described [16 (link), 17 (link)]. 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS.
7
Cultivating and Analyzing NSCLC Cell Lines
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The A549 and NCI-H1299 human NSCLC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibico; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C in a humidified atmosphere containing 5% CO2. Antibodies against RKIP (cat. no. 13006), STAT3 (cat. no. 9139), phosphorylated (p)STAT3 (cat. no. 9145), ERK (cat. no. 9102) and pERK (cat. no. 4370) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and β-actin (cat. no. 7210) antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
8
Protective Effects of FO and LM on PA-Induced Lipid Accumulation
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HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% FBS and 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibico, Thermo Fisher Scientific, San Jose, CA, USA) at 37 °C and 5% CO2. The PA solution was produced as previously described, albeit with a small change [27 (link)]. After being dissolved in 1 M NaOH and heated to 70 °C, 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) was immediately mixed with PA and diluted in DMEM. The HepG2 cells were divided into five groups: (1) NC, where the cells were grown in DMEM with 0.5% BSA supplement as control group; (2) PA, where the cells were incubated with 150 μM PA solution promoting lipid accumulation; (3) PA + FO, where the cells were pretreated with FO at 1 mg/mL for 6 h followed by further stimulation with PA at 150 μM for 18 h; (4) PA + LM, where the cells were pretreated with LM at 1 μg/mL for 6 h and then further stimulated with PA at 150 μM for 18 h; and (5) PA + LM + FO, where the cells were treated with FO combined with LM for 6 h followed by incubation with PA till 24 h.
9
VEGF Inhibition Efficiency Assay
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The anti‐VEGF efficiency of recombinant proteins was compared using a HEK293 reporter gene‐based assay, which has been previously described.
27 (link) Briefly, HEK293 cells were transfected with VEGFR2 and NFAT‐RE‐luc2p genes, and activation of the NFAT transcription factor, which occurs upon triggering of the VEGF‐VEGFR2 pathway, promotes the expression of the Luciferase gene, which can be detected using luciferin substrate. The cells were sub‐cultured in 1% FBS DMEM (Gibico, Thermo Fisher Scientific, USA) at a density of 5 × 105 cells/mL and were added to a 96‐well white plate (Costar, Corning, Inc., USA) at a volume of 80 μL to incubate overnight at 37°C. The test protein was serially diluted three times from 100 nM using 1% FBS DMEM, and the same volume (60 μL) of 80 ng/mL VEGF165 in 1% FBS DMEM was added to each dilution. The mixtures were incubated at 37°C for 30 min, and then 20 μL of the mixture was added to the cells for 6 h of incubation at 37°C until luminescence measurement (Firefly Glo Luciferase Reporter Gene Assay Kit, YEASEN, Shanghai, China).
27 (link) Briefly, HEK293 cells were transfected with VEGFR2 and NFAT‐RE‐luc2p genes, and activation of the NFAT transcription factor, which occurs upon triggering of the VEGF‐VEGFR2 pathway, promotes the expression of the Luciferase gene, which can be detected using luciferin substrate. The cells were sub‐cultured in 1% FBS DMEM (Gibico, Thermo Fisher Scientific, USA) at a density of 5 × 105 cells/mL and were added to a 96‐well white plate (Costar, Corning, Inc., USA) at a volume of 80 μL to incubate overnight at 37°C. The test protein was serially diluted three times from 100 nM using 1% FBS DMEM, and the same volume (60 μL) of 80 ng/mL VEGF165 in 1% FBS DMEM was added to each dilution. The mixtures were incubated at 37°C for 30 min, and then 20 μL of the mixture was added to the cells for 6 h of incubation at 37°C until luminescence measurement (Firefly Glo Luciferase Reporter Gene Assay Kit, YEASEN, Shanghai, China).
10
Establishing Human Cell Lines for EGFR Knockdown
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Human retinoblastoma cell line Weri-Rb-1 and Human Embryo Kidney cell line 293T were purchased from Cell Bank of the Chinese Academy of Sciences and American Type Culture Collection, respectively. Both cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Hyclone, GE Healthcare) supplemented with 10% fetal calf serum (Hyclone, GE Healthcare) and antibiotics (GIBICO, ThermoScientific). Lentiviral vectors carrying EGFR-specific shRNA (TL320326) or negative control shRNA (TR30021) were purchased from origin and lentiviral particles were propagated on 293T cell line according to the manufacturer’s instructions.
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