Human THP-1 cells were seeded in each well of a 6-well upper Transwell insert (0.4 μm microporous membrane, Corning) and placed in a separate 6-well plate containing PMA for polarization. On the day before macrophage polarization was completed, HCCLM3 and Huh7 cells were seeded in fresh 6-well lower Transwell plates and were allowed to attach overnight. After the polarization was completed, THP-1 macrophages in the upper Transwell inserts were carefully washed with phosphate-buffered saline (KeyGen Biotechnology), and each upper Transwell insert was transferred to a 6-well lower Transwell plate containing hepatoma cells. The polarized macrophages and hepatoma cells were then cocultured in the RPMI 1640 medium for 24 h.
Phosphate buffered saline (pbs)
Manufactured by Keygen Biotech
Sourced in China
PBS, or Phosphate-Buffered Saline, is a commonly used buffer solution in laboratory settings. It is a balanced salt solution that maintains a stable pH and ionic environment, providing a suitable medium for various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Keygen Biotech (4 mentions)
Penicillin streptomycin, by Keygen Biotech (3 mentions)
Rpmi 1640 medium, by Keygen Biotech (2 mentions)
High glucose dmem, by Keygen Biotech (2 mentions)
Fetal bovine serum (fbs), by Absin (2 mentions)
Milli q water purification system, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Keygen Biotech (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Jem 1011, by JEOL (2 mentions)
Dulbecco modified eagle medium (dmem), by Keygen Biotech (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Hypoxia oxidative stress detection kit, by Enzo Life Sciences (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin mixture, by Keygen Biotech (1 mentions)
43 protocols using phosphate buffered saline (pbs)
1
Transwell Co-culture Polarized Macrophages
2
Investigating Tumor-Endothelial Cell Interactions
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Human umbilical vein endothelial cells (HUVECs) and mouse breast cancer cells (4T1) were supplied by the Shanghai Institute of Cell Biology (Shanghai, China). Roswell Park Memorial Institute 1640 (RPMI-1640) medium and phosphate-buffered saline (PBS, pH = 7.4, 6.8, 5.5) were purchased from KeyGen BioTech (Nanjing, China). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). The hypoxia/oxidative stress detection kit was obtained from Enzo Life Sciences (New York, United States). pHrodo™ Rea AM (Invitrogem™, United States) LysoTracker Deep Red was purchased from Thermo Fisher Scientific Incorporated (America). All cells used in this work were incubated in RPMI-1640 medium supplemented with 10% FBS at 37°C with 5% CO2.
3
Isolation of Rat Bone Marrow Stromal Cells
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The 2-week-old male Sprague Dawley (SD) rats were used as donors to culture primary rBMSCs. Rats were anesthetized by inhalation of 3% isoflurane and then euthanized by intraperitoneal injection of sodium pentobarbital (70 mg/kg). The long bones were obtained by removing the skin of the two hind legs and dissecting them at the hip, knee, and ankle joints. Femur and tibia with residual tissue removed were rinsed with phosphate-buffered saline (PBS, Keygen Biotech, China). The prepared femur and tibia were immersed in DMEM/F12 medium (Keygen Biotech, China) with 10% fetal bovine serum (FBS, Sigma, USA) and 1% penicillin-streptomycin mixture (Keygen Biotech, China). Bone marrow was collected by continuous rinsing with DMEM/F12 medium after insertion of a 22-gauge needle into the midsection of the femur and tibia. Collect the bone marrow cell suspension in a sterile 15 mL centrifuge tube and then centrifuge at 1000 rpm for 3 min. Resuspend the pellet culture medium. The suspended cells were cultured in a T25 cm2 flask and placed in a 37°C incubator containing 5.0% CO2. Replace the medium every 3 days. All experiments used BMSCs at passage 3.
4
PEG Conjugation and Characterization
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Maleimide-functionalized PEG (mPEG-Mal) (Mw ≈ 2 kDa) and t-Boc-NH-PEG-Mal (Mw ≈ 2 kDa) were purchased from JenKem Technology Co. Ltd (Beijing, China). Na3C6H5O7·2H2O, CuCl2, Na2S, cysteamine, 4,4′-diamino-2,2′-bipyridine (DABPY), tetra-n-butylammonium bromide, furfurylamine were purchased from Energy Chemical (Shanghai, China). MnBr(CO)5, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), myoglobin, and folic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All reagents used were of analytical grade. The water used in all experiments was deionized (DI) water with a resistivity of 18.2 MΩ cm. The molecular weight cutoff of all dialysis bags used in the dialysis process was of 5 kDa. High-glucose DMEM containing 1% penicillin/streptomycin, phosphate-buffered saline (PBS), and trypsin was obtained from KeyGen BioTech Co. Ltd. (Jiangsu, China). Fetal bovine serum was purchased from Absin Bioscience Inc. (Shanghai, China).
5
Isolation and Characterization of Granulosa Cells
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During oocyte retrieval, FF was collected from follicles with a diameter ≥ 16 mm measured on the retrieval day, then immediately separated by centrifugation at 700×g for 5 min at room temperature. The precipitates were suspended in left 2 ml of FF and gently layered into 3 mL of 50% lymphocyte separation medium (Solarbio Science and Technology Corporation, Beijing, China). After centrifugation at 700×g for 10 min at room temperature to remove red blood cells and debris, GCs layered at the interface of the gradient were collected and washed twice with 5 mL of phosphate-buffered saline (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, Jiangsu, China). The residual red blood cells were further removed using red blood cell lysis buffer (Solarbio Science and Technology Corporation). GCs from each patient were collected separately and considered as one sample. One portion of GCs was immediately examined intracellular ROS levels, mitochondrial membrane potential (MMP), and apoptosis; the remaining GCs were stored at − 80 °C refrigerator.
6
Antimicrobial Activities of TCMs
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Polygonum cuspidatum Sieb. et (P.C.), Fagopyrum dibotrys (D. Don), Sanguisorba officinalis L., Agrimonia pilosa Ledeb., Hedyotis diffusa Willd, Rheum palmatum L., and Geranium wilfordii Maxim were provided by Bozhou Pharmaceutical Co Ltd (Bozhou, People’s Republic of China). These TCMs were authenticated by Professor Shihui Qian, at the Jiangsu Provincial Academy of Chinese Medicine. Silver nitrate, phosphoric acid, and trisodium citrate were obtained from Sigma-Aldrich (St Louis, MO, USA). Strains of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus were obtained from the Institute of Microbiology, Chinese Academy of Sciences (Beijing, People’s Republic of China). Beef extract-peptone medium and phosphate-buffered saline were sourced from Nanjing KeyGEN Biotech Co Ltd (Nanjing, People’s Republic of China). Water in this study was provided by a Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA). Other chemicals and reagents were of analytical grade.
7
Evaluating IgY Protein Release in vitro
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For assessment of the IgY protein release property in vitro, 10 mg of freeze-dried IgY@ACP powder was dissolved in 10 ml of phosphate-buffered saline (PBS; Keygen Biotech, Nanjing, China) (pH = 7.4) and placed in a temperature oscillator with constant shaking (37°C, 180 rpm). At the given time points (5, 10, 20, and 40 min; 1, 2, 3, 6, 12, 18, 24, 30, 42, and 48 h), a certain volume of solution was extracted and centrifuged to collect the supernatant, which was then replaced with an equal volume of fresh PBS solution. The previously mentioned supernatant was incubated with a BCA protein assay kit (Beyotime, Shanghai, China) in a 96-well plate (Corning, USA) for 2 h in the dark to detect protein concentrations at corresponding time points. The OD value of each well at a wavelength of 562 nm was observed with a microplate reader (Biotek, Winooski, Vermont, USA). The protein concentrations of the samples at corresponding time points were calculated according to the standard protein curve equation and the sample volume used, and finally, the cumulative protein release rate curve was generated.
8
Extraction and Characterization of A. herba
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A. herba was purchased from Bozhou Huqiao Pharmaceutical (Bozhou, People’s Republic of China), and was authenticated by Shihui Qian, a professor at the Jiangsu Provincial Academy of Chinese Medicine. Aluminum chloride, anhydrous sodium carbonate, silver nitrate, and phosphoric acid were provided by Nanjing Chemical Reagent (purity >99.8%, analytical reagent; Nanjing, People’s Republic of China). Folin-Ciocaileu reagent was obtained from Nanjing Duly Biotech (Nanjing, People’s Republic of China). Rutin and gallic acid reference substances (purity >99.5%) were purchased from the National Institutes for Food and Drug Control (Beijing, People’s Republic of China). Trisodium citrate was provided by Kunshan Jincheng Reagent (Kunshan, People’s Republic of China). MTT, dimethyl sulfoxide (DMSO), Roswell Park Memorial Institute (RPMI) 1640 medium, trypsin, and phosphate-buffered saline were provided by Nanjing KeyGen Biotech (Nanjing, People’s Republic of China). Fetal bovine serum (FBS) was bought from Zhejiang Tianhang Biological Technology (Hangzhou, People’s Republic of China). Water in this study was provided by the Milli-Q water-purification system (EMD Millipore, Billerica, MA, USA). All other chemicals and solvents were of analytical or reagent grade.
9
Comprehensive Reagent Procurement for Multifaceted Research
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Folin-Ciocalteu reagent (FCR) and tripyridyltriazine (TPTZ) were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). The following chemicals were obtained from KeyGen Biotech (Nanjing, China): 2, 2-diphenyl-1-picryl-hydrazyl (DPPH); 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT); 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA); lipopolysaccharide (LPS, from Escherichia coli O111:B4); phosphate-buffered saline (PBS); N-acetyl-L-cysteine (NAC); BCA protein quantitative kit; and nicotinamide mononucleotide (NMN). Fetal bovine serum (FBS), high-glucose Dulbecco’s modified Eagle’s medium (DMEM), enhanced chemiluminescence (ECL) detection kit, and bovine serum albumin (BSA) were obtained from Thermo Scientific (Rockford, IL, USA). The primary and secondary antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA). All other chemicals and solvents were of analytical grade and obtained from Guanghua Sci-tech (Shantou, China).
10
Cell Culture Reagent Preparation
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DMSO was purchased from Sigma-Aldrich (Shanghai, China). An aliquot of 0.25% Tripsin-EDTA, Trypan blue, and PBS (calcium and magnesium free) were purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). GIBCO DMEM was produced by Invitrogen (Grand Island, NY). Fluo-3 AM (Calcium ion fluorescence probe, 5 mM) was purchased from Beyotime Institute of Biotechnology (Haimen, China). Water was deionised using the Milli-Q-Plus ultra-pure water system (Millipore, Milford, MA).
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