Cryopreserved PBMCs were thawed rapidly in a water bath at 37°C, added to R-10 culture media, washed and resuspended in RPMI 1640 medium supplemented with 10% Heat Inactivated Foetal Calf Serum (HI-FCS; SAFC, Brooklyn, Australia), 2-mercaptoethanol (Sigma-Aldrich, Castle Hill, Australia), glutamax (Life Technologies, Melbourne, Australia), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Life Technologies), antibiotic-antimycotic (Life Technologies) and sodium pyruvate (Life Technologies) at a density of 2x106 PBMCs/mL. PBMCs were plated in sterile polypropylene round-bottomed 96-well plates (Corning Costar, Lowell, USA) in a final volume of 250μL, with 5x105 PBMCs per well. PBMCs were stimulated with 0.9% (w/v) NaCl (Baxter, Old Toongabbie, Australia) as a negative control, 10μg/mL TT (kindly provided by GSK, Rixensart, Belgium) or 2.5μg/mL Staphylococcal Enterotoxin B (SEB; Sigma-Aldrich) as a positive control. PBMCs were stimulated for 48 hours in a 37°C humidified incubator (Thermo Scientific, Waltham, USA) with 5% CO2, and 3μg/mL Brefeldin A solution (eBioscience, San Diego, USA) was added to wells for the final 16 hours of culture. Following culture, cells were harvested by gentle pipetting and washed prior to staining for expression of surface and intracellular markers.
37 c humidified incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 37 °C humidified incubator is a piece of laboratory equipment designed to maintain a consistent temperature of 37 degrees Celsius and a humid environment. It is used to create optimal conditions for the growth and maintenance of cell cultures, tissues, and other biological samples that require a controlled temperature and humidity setting.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Raw264, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Baxter (1 mentions)
Staphylococcal enterotoxin b, by Merck Group (1 mentions)
Brefeldin a solution, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Recombinant il 2, by ProSpec (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
L929 cell, by American Type Culture Collection (1 mentions)
Lx 2 cells, by American Type Culture Collection (1 mentions)
Aml12 cells, by American Type Culture Collection (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Aspc 1, by Procell (1 mentions)
Miapaca 2, by Procell (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
8 protocols using 37 c humidified incubator
1
PBMC Stimulation and Cytokine Analysis
2
PBMC Stimulation for Cytokine ELISpot
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Methods were adapted from a method previously described for Pneumococcal antigens [17 (link)]. Briefly, thawed PBMCs were re-suspended in RPMI 1640 medium supplemented with 10% HI-FCS (SAFC), 2-mercaptoethanol (Sigma-Aldrich), glutamax (Life Technologies), HEPES (Life Technologies), antibiotic-antimycotic (Life Technologies) and sodium pyruvate (Life Technologies) at a density of 1x106/mL. Two-hundred micro-litre aliquots were plated onto polypropylene round-bottomed 96-well culture plates (Corning Costar). Thirty micro-litres of stimulation mix comprising; CpG-2006 ODN (TIB MOLBIOL, Berlin, Germany), recombinant IL-2 (Prospec-Tany, Ness-Ziona, Israel) and recombinant IL-15 (Prospec-Tany) was added to each well to give a final concentration of 3μg/mL, 10ng/mL and 10ng/mL respectively. Cells were stimulated for 6 days in a 37°C humidified incubator (Thermo Scientific) with 5% CO2 prior to commencement of ELISpot.
3
Cell Culture Protocol for Diverse Cell Lines
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A non-tumorigenic mouse hepatocyte cell line AML-12 cells, a murine macrophage cell line Raw-264.7 cells, a human hepatic stellate cell line LX2 cells, and a mouse fibroblast cell line L929 cells were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). AML-12 cells were cultured in DMEM/F12 medium containing 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, 40 ng/mL dexamethasone, 10% FBS, and 1% penicillin/streptomycin in a 37°C humidified incubator (Thermo, USA) with 5% CO2 and 95% air.
4
Murine and Human Cell Lines for In Vitro and In Vivo Studies
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A murine macrophage cell line RAW264.7, a human non-small cell lung cancer cell line A549, a A549/GFP (GFP, green fluorescent protein) human non-small cell lung cancer cell line stably expressing GFP, a human lung fibroblast cell line HLF were purchased from the American Tissue Culture Collection. All cells were kept in a 37 °C humidified incubator (Thermo, U.S.A) with 5% CO2. Female BALB/c nude mice (four weeks old) were purchased from the Laboratory Animal Center, Sun Yat-sen University (Guangzhou, China) and kept under SPF conditions, with ready access to standardized food and water. All the animal experiments conducted were strictly observed the Guiding Principles for the Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University.
5
Cell Culture Conditions for Pancreatic Cancer
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ASPC-1 and MIA-PaCa2 were purchased from Procell (Wuhan, China). All cell lines were authenticated. ASPC-1 was cultured in 1640 (VivaCell, Shanghai, China) with 10% 30070 (Hyclone, Logan, UT, USA), and MIA-PaCa2 was cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; VivaCell, Shanghai, China) with 10% 30070 and 3% horse serum (Gibco, Grand Island, New York City, NY, USA). Both of the cell lines were cultured in a 37 °C humidified incubator (Thermo, Waltham, MA, USA) with a 5% CO2 environment.
6
Modulating RGS1 and miR-376b-3p in Osteosarcoma
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MG63 and U2OS cell lines (purchased from ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium containing high-glucose (DMEM-HG), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all purchased from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured in a 37 °C humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO2. For transfection, a lentivirus-packaged RGS1 vector, short hairpin (sh) RNAs, miR-negative control (NC), miR-376b-3p mimic, Sponge NC, and sponge miR-376b-3p were purchased from Hanyin Biotechology (Shanghai, China) (10 (link)). Cell transfection was applied according to the manufacturer’s instructions. The levels of RGS1 protein in both RGS1 stably over-expressed and stably silenced cells were verified every 2 weeks with western blotting.
The sequence of the shRNAs and mimics were as follows: sh negative control (NC), 5'-CAGUACUUUUGUGUAGUACAAA-3'; shRGS1 #1, 5'-GCATATCTAAGATCTATGATC-3'; shRGS1 #2, 5'-GGGATGAAATCGTCCAAGTCC-3'; miR-NC, 5'-AAGAAUCAGGUUUUUCCAUGUU-3'; miR-376b-3p (mimic), 5'-AUCAUAGAGGAAAAUCCAUGUU-3'; sponge NC, 5'-AUCUUCGAAAUUCCUCUAUGUU-3'; and sponge miR-376b-3p, 5'-AACAUGGAUUUUCCUCUAUGAU-3'.
The sequence of the shRNAs and mimics were as follows: sh negative control (NC), 5'-CAGUACUUUUGUGUAGUACAAA-3'; shRGS1 #1, 5'-GCATATCTAAGATCTATGATC-3'; shRGS1 #2, 5'-GGGATGAAATCGTCCAAGTCC-3'; miR-NC, 5'-AAGAAUCAGGUUUUUCCAUGUU-3'; miR-376b-3p (mimic), 5'-AUCAUAGAGGAAAAUCCAUGUU-3'; sponge NC, 5'-AUCUUCGAAAUUCCUCUAUGUU-3'; and sponge miR-376b-3p, 5'-AACAUGGAUUUUCCUCUAUGAU-3'.
7
Murine and Human Cell Lines in Cancer Research
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A murine macrophage cell line RAW264.7, a human ovarian cancer cell line SKOV3 and a Chinese hamster ovary cell line CHO were purchased from the American Tissue Culture Collection. A stable green fluorescent protein (GFP)-expressing SKOV3 cell line was a gift from Qi Wang, Guangxi Medical University (Guangxi, China). All cells were incubated in a 37 °C humidified incubator (Thermo, USA) with 5% CO2. Female BALB/c nude mice (four-week old) were purchased from the Laboratory Animal Center, Sun Yat-sen University (Guangzhou, China) and kept under specific pathogen-free (SPF) conditions, with ready access to standardized food and water. All animal experiments were strictly observed under the Guiding Principles for the Use of Laboratory Animals approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University.
8
Establishment and Characterization of Luciferase-Tagged Human Liver Cancer Cell Lines
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The human HCC cell line MHCC97L, which was tagged with a luciferase reporter gene, was gifted by Professor Man Kwan from the Department of Surgery, The University of Hong Kong. The HLF cell line was purchased from Japanese Collection of Research Bioresources cell bank. Both cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 4.5g/L glucose, 10% foetal bovine serum (Gibco, MA, USA) and 1% penicillin/streptomycin (Gibco, MA, USA)); and they were kept in a 37 °C humidified incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA) under 5% CO 2 . The DNA plasmid encoding pBABE-puro LEF1 was gifted by Joan Massague (plasmid #27023; Addgene Inc., Cambridge, MA); and the DNA plasmid expressing pAd/WT1-IRES-nAmCyan was gifted by Edward McCabe (plasmid #29756, Addgene). Mangiferin was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The stock solution of Mangiferin (100 mg/mL) was prepared in DMSO (Sigma-Aldrich Co., St. Louis, M.O., USA) and dH 2 O (1:9, v/v). For animal administration, Mangiferin (50 mg/kg) was prepared in 0.4% of sodium carboxyl methyl cellulose (Sigma-Aldrich Co., St. Louis, M.O., USA).
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