Cell culture dishes, plates, centrifuge tubes, and other plastic ware were purchased from BD Biosciences (Lincoln Park, NJ); Dulbecco modified Eagle medium (DMEM), Ham F-12, amphotericin B, and gentamicin were from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). RNeasy Plus Mini RNA extraction kit from Qiagen (Valencia, CA); Ready-To-Go-Primer First-Strand Beads were from GE Healthcare (Piscataway, NJ); TaqMan gene expression assays and real-time PCR master mix were from Applied Biosystems (Foster City, CA). DCFDA—Cellular Reactive Oxygen Species Detection Assay Kit, rabbit polyclonal antibody against human malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE), aconitase-2, glutathione peroxidase-1 (GPX1), and mouse monoclonal antibody against superoxide dismutase-1 (SOD1) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibody against human heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2) were from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody was from BioLegend (San Diego, CA). Fluorescein Alexa-Flour 488-conjugated secondary antibodies (donkey anti-rabbit, or goat anti-mouse IgG) were from Molecular Probes (Eugene, OR).
Centrifuge tube
Manufactured by BD
Sourced in United States
Centrifuge tubes are laboratory equipment used to separate different components of a liquid mixture by centrifugal force. They are typically made of plastic or glass and are designed to withstand high speeds and forces during the centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
Cell culture dishes, by BD (3 mentions)
Gentamicin, by Thermo Fisher Scientific (3 mentions)
Rneasy plus mini rna extraction kit, by Qiagen (3 mentions)
Ready to go primer first strand beads, by GE Healthcare (3 mentions)
Real time pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Falcon tissue culture flasks, by BD (3 mentions)
Vybrant cell labelling solutions, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Gemini Bio (2 mentions)
L carnitine, by Merck Group (2 mentions)
Betaine, by Merck Group (2 mentions)
Erythritol, by Merck Group (2 mentions)
Amphoteracin b, by Thermo Fisher Scientific (2 mentions)
Saos 2 osteosarcoma cells, by American Type Culture Collection (2 mentions)
Glutathione peroxidase 1 gpx1, by Abcam (1 mentions)
Dcfda cellular reactive oxygen species detection assay kit, by Abcam (1 mentions)
Hmox1, by Santa Cruz Biotechnology (1 mentions)
Cyclooxygenase 2 cox 2, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by BioLegend (1 mentions)
27 protocols using centrifuge tube
1
Oxidative Stress Biomarkers Analysis
2
Cell Labeling Techniques for Experiments
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For experiments performed at MSKCC, all culture media including M199, α-MEM, Trypsin (0.25%)-EDTA (0.02%) and PBS, as well as Penicillin (10,000 U/mL)-Streptomycin (10,000 μg/mL) concentrate solution were prepared and supplied by the Memorial Sloan-Kettering Cancer Centre Culture Media Core Facility (New York, NY, USA). Amphotericin B was purchased from Life Technologies (Grand Island, NY, USA). Gelatin was from TJ Baker Inc. (Philipsburgh, NJ, USA). Bovine serum albumin was from Gemini Bioproducts (West Sacramento, CA, USA). Falcon tissue culture flasks and centrifuge tubes were purchased from BD Biosciences (Two Oak Park, Bedford, MA). Nunc plastic culture well coverslips were from ThermoFisher, Waltham, MA, USA). The lipophilic fluorescent probes DiD (excitation 644 nm, emission 665 nm) and DiO (excitation 484 nm, emission 501 nm) Vybrant cell labelling solutions were from Molecular Probes, Life Technologies (Grand Island, NY, USA), and were used in all experiments at both the MSKCC and CMPRU.
3
Inflammatory Cytokine Assay Protocol
4
Culturing Human Dermal Fibroblasts and Osteosarcoma Cells
5
Blood Separation and Cryopreservation Protocol
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Within 2–4 h after blood donation, up to 10 ml anti-coagulated blood was separated in centrifuge tubes (BD Biosciences, MD, USA) prefilled with 5 ml Ficoll Paque (GE Healthcare, Uppsala, Sweden), according to the instructions of the manufacturer. After being centrifuged at 3500 rpm for 10 min at 4 °C, plasma supernatant was harvested and stored at − 80 °C until further analysis. The enriched cell fraction containing PBMCs was harvested and washed twice in 10 ml phosphate-buffered saline (PBS). Cell pellets were shock-frozen for cryopreservation at − 80 °C until further usage.
6
Fluorescent Labeling of Cell Lines
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All culture media including M199, α-MEM, M5, Trypsin (0.25%)-EDTA (0.02%) and PBS, as well as Penicillin (10,000 U/ml)-Streptomycin (10,000 μg/ml) concentrate solution were prepared and supplied by the Memorial Sloan-Kettering Cancer Centre Culture Media Core Facility (New York, NY). Amphoteracin B was purchased from Life Technologies (Grand Island, NY). Gelatin was from TJ Baker Inc (Philipsburgh, NJ). Bovine serum albumin was from Gemini Bioproducts (West Sacramento, CA). Falcon tissue culture flasks and centrifuge tubes were purchased from BDBiosciences (Two Oak Park, Bedford, MA). HDF were from The Coriell Institute (Camden, NJ). SAOS-2 osteosarcoma cells were from the American Type Culture Collection (VA, USA). A673 osteosarcoma and H3122 lung carcinoma cells were from the collection at the Memorial Sloan Kettering Cancer Center. The lipophilic fluorescent probes DiD (excitation 644nm, emission 665nm) and DiO (excitation 484nm, emission 501nm) Vybrant cell labelling solutions were from Molecular Probes, Life Technologies (Grand Island, NY). DAPI was provided by the FACS core facility at Memorial Sloan Kettering Cancer Center.
7
Quantification of MMP Expression and Activity
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Cell culture dishes, plates, centrifuge tubes, and other plastic ware were purchased from BD Biosciences (Lincoln Park, NJ). Dulbecco modified Eagle medium (DMEM), Ham F-12, amphotericin B, and gentamicin were from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). L-carnitine, erythritol, and betaine were from Sigma-Aldrich (St. Louis, MO). The RNeasy Plus Mini RNA extraction kit from Qiagen (Valencia, CA). The Ready-To-Go-Primer First-Strand Beads were from GE Healthcare (Piscataway, NJ). The TaqMan gene expression assays and real-time PCR master mix were from Applied Biosystems (Foster City, CA). The MMP antibodies were from Sigma-Aldrich (St. Louis, MO). The ready zymogram gels were from Bio-Rad (Hercules, CA).
8
Anti-inflammatory Activity Evaluation
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For the evaluation of anti-inflammatory activity, Raw 264.7 cells were cultured in a 6 well plate and treated with 3 different culture conditions viz., LPS (2ug/ml), LPS (2µg/ml)+Turmesac® (50μg/ml) and untreated without any treatment. Briefly, the cells were pre stimulated with 2 µg/ml of LPS for 3 h to induce inflammation and following stimulation, the cells were either treated with 50µg/ml of Turmesac® or LPS stimulated alone (negative control) with 2 ml DMEM medium. Cells were incubated for 24 h and harvested into centrifuge tubes (BD Biosciences) and centrifuged at 300 × g for five minutes in a Remi: R-8 °C centrifuge and were washed twice with D-PBS. The pelleted cells were incubated at room temperature with 0.5 ml BD Cytofix/Cytoperm for 10 min and washed with 0.5% Bovine Serum Albumin solution (1x PBS and 0.1% sodium azide). Cells were incubated with 20μl of PE-Mouse Anti Human interleukin 8 (IL-8) antibody/Anti Human IL-12 separately for 30 min in the dark at 25 °C and expression measured using a BD FACS Calibur flow cytometer (BD Biosciences) and data analyzed by Cell Quest Pro software version 6.
9
Cytokine Expression in LPS-Stimulated Macrophages
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Raw 264.7 cells were grown in a 6-well plate under 3 distinct culture conditions: LPS (2 µg/mL), LPS (2 µg/mL) +Turmimax ® (50 µg/mL), and untreated, which received no treatment. Briefly, the cells were first stimulated with 2 µg/mL of LPS for 3 h to generate inflammation and then either treated with 50 µg/mL of Turmimax ® or stimulated with LPS alone (negative control) in 2 mL of DMEM media. Cells were cultured for 24 h, collected, and then centrifuged at 300xg for five minutes in a Remi: R-8°C centrifuge using centrifuge tubes from BD Biosciences. After that, D-PBS was used to wash them twice. The pelleted cells were then rinsed with a 0.5% bovine serum albumin solution (1 x PBS and 0.1% sodium azide) and incubated at room temperature with 0.5 mL of BD Cytofix/Cytoperm for 10 min. Cell Quest Pro software version 6 was used to analyze the data after the cells were incubated with 20 µL of a PE-mouse anti-human interleukin 8 (IL-8) antibody and an anti-human IL-12 antibody separately for 30 min in the dark at 25°C. Expression was then measured using a BD FACS Caliber flow cytometer (BD Biosciences).
10
Acute Restraint Stress Procedure in Mice
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Acute restraint stress was performed using clear 50ml conical vials (Falcon Centrifuge Tubes) that had ten 5mm holes for ventilation. Animals were placed individually into their own tubes and restrained for 2 hours in a biosafety cabinet with overhead lights. Each tube was placed flat in an open box (120mm X 80mm X 50mm) to prevent minimize movement of the tube. Animals could hear and smell one another but not see each other during the procedure. Mice in nonstressed groups were briefly handled in another room during the restraint stress procedure. Following the procedure, all animals were returned to their homecage where they remained for 30 days. All animals were handled each week briefly for cage changes.
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