Liver homogenates were prepared by mincing and homogenizing approximately 100 mg of liver tissue in 1 mL cold PBS buffer with a steel homogenizer. The homogenate was centrifuged using Thermo Scientific centrifuge (Legend Micro 17 R) at 4000 rpm and 4 °C for 25 min. The resultant supernatant was used for the determination of enzymes’ activities [12 (link)].
Legend micro 17r
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Legend Micro 17R is a high-performance, compact refrigerated microcentrifuge from Thermo Fisher Scientific. It is designed for a wide range of applications, including sample preparation, DNA/RNA extraction, and protein purification. The Legend Micro 17R features a maximum speed of 17,000 rpm and a maximum relative centrifugal force (RCF) of 24,600 × g, making it suitable for a variety of sample volumes and types. The centrifuge is equipped with a brushless, maintenance-free motor and a robust, quiet operation.
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Lab products found in correlation
Zetasizer nano zs90, by Malvern Panalytical (3 mentions)
Mastersizer 3000, by Malvern Panalytical (1 mentions)
E2695 2998, by Waters Corporation (1 mentions)
D 37520, by Martin Christ (1 mentions)
Uv 2450, by Shimadzu (1 mentions)
Rodent diet d12492, by Research Diets (1 mentions)
D12492, by Research Diets (1 mentions)
Zoletil, by Virbac (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Zen lite 2012, by Zeiss (1 mentions)
10 protocols using legend micro 17r
1
Liver Homogenization and Enzyme Extraction
2
Measuring Water Holding Capacity and Cooking Loss of Gel Samples
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The water WHC of the gel samples were measured by centrifugation (Legend Micro 17R, Thermo Fisher Scientific, Waltham, MA, USA) with a slight modification [14 (link)]. First, before centrifugation, the weight of the sample was recorded as m1. The mass of the sample after centrifugation was then recorded as m2. The WHC was calculated as follows:
The cooking loss was measured according to Cao & Xiong [15 (link)]. m1 is the weight of each gel sample. m2 is the weight of the sample after heating. The cooking loss was calculated as follows:
The cooking loss was measured according to Cao & Xiong [15 (link)]. m1 is the weight of each gel sample. m2 is the weight of the sample after heating. The cooking loss was calculated as follows:
3
Characterization of Fasudil-Loaded Liposomes
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CAR–liposomes
were characterized for size, polydispersity
index (PDI), zeta potential, and entrapment efficiency of fasudil
according to our previously published method.22 (link) To measure the size and zeta potential, 20 μL of liposomes
was taken in an Eppendorf tube and diluted to 1000 μL with PBS
and water. Both the size and zeta potential of the liposomes were
measured by Nano ZS90 Zetasizer (Malvern Instruments Ltd., Worcestershire,
U.K.). To quantify entrapped fasudil, 20 μL of liposomes was
placed in an Eppendorf tube, mixed with 980 μL of methanol,
sonicated for 15 min, and centrifuged at 17000g (Legend
Micro 17R, Thermo Scientific) for 15 min, and the absorbance of the
supernatant was measured at 320 nm in a UV spectrophotometer (HP 8453A,
Olis Inc.) The fasudil encapsulation efficiency was calculated using
the formula L/T × 100 (where L = the amount of fasudil incorporated into liposome, T = total amount of fasudil).
were characterized for size, polydispersity
index (PDI), zeta potential, and entrapment efficiency of fasudil
according to our previously published method.22 (link) To measure the size and zeta potential, 20 μL of liposomes
was taken in an Eppendorf tube and diluted to 1000 μL with PBS
and water. Both the size and zeta potential of the liposomes were
measured by Nano ZS90 Zetasizer (Malvern Instruments Ltd., Worcestershire,
U.K.). To quantify entrapped fasudil, 20 μL of liposomes was
placed in an Eppendorf tube, mixed with 980 μL of methanol,
sonicated for 15 min, and centrifuged at 17000g (Legend
Micro 17R, Thermo Scientific) for 15 min, and the absorbance of the
supernatant was measured at 320 nm in a UV spectrophotometer (HP 8453A,
Olis Inc.) The fasudil encapsulation efficiency was calculated using
the formula L/T × 100 (where L = the amount of fasudil incorporated into liposome, T = total amount of fasudil).
4
Characterization of Liposomal Formulations
5
Characterization of CAR-Conjugated Liposomes
6
Analytical Techniques for Pharmaceutical Development
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The instruments used in this study included a high-performance liquid chromatograph (e2695-2998, Waters, Milford, MA, USA), a digital mixer (RW20, IKA, Staufen, Germany), a lyophilizer (D-37520, Christ, Osterode, Germany), a polarizing microscope (BK-POL, Aote Optical Instrument, Chongqing, China), a thermostatic oscillator (SHA-C, GY2016-SW, Changzhou Guoyu, Changzhou, China), a digital rotary viscometer (NDJ-8S, Fangrui, Shanghai, China), laser-diffraction particle size analyzers (Mastersizer 3000, Malvern, Worcestershire, UK), a moisture analyzer (Metrohm, Herisau, Switzerland), heat and accelerated stability chambers (Yongsheng, Chongqing, China), a UV spectrophotometer (UV 2450, Shimadzu, Kyoto, Japan), an ultra-high-performance liquid chromatograph (e2695-2998, Waters, Milford, MA, USA), an LC-MS/MS system (6460, Agilent, Santa Clara, CA, USA), and a refrigerated centrifuge (Legend Micro 17R, Thermo, Waltham, MA, USA).
7
Obesity Induction in C57BL/6 Mice
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The experiments were approved by the Animal Ethics Committee of the Dongshin University (Approval No. 2019-07-02). To evaluate the effects of obesity, C57BL/6 mice were purchased from SamTako BioKorea (Osan, Korea) and acclimatized for 7 days. According to the type of diet, 12 mice were divided into two groups (Research Diet, Inc. Product #D12492, New Brunswick, NJ, USA) and given tap water for 8 weeks: (1) control group (n = 6); (2) obesity group with a HFD (D12492 Rodent Diet with 60% fat, 20% protein, 20% carbohydrate, Research Diets, Inc., New Brunswick, NY, USA) (n = 6). Ingredients used for the control and obesity group diets are provided in Tables S5 and S6 .
The body weights of the mice were recorded once per week. All mice were anesthetized with 50 mg/kg Zoletil (Virbac, TX, USA). To determine metabolites in the serum, the collected whole blood was centrifuged at 12,000 rpm using a Legend micro 17-R (Thermo Fisher Scientific, Waltham, MA, USA). TCHO-P III and GLU-P III levels were measured. Mice were euthanized using Zoletil over-dosing, then the abdominal fat tissues were collected and the weights of these tissues were measured.
The body weights of the mice were recorded once per week. All mice were anesthetized with 50 mg/kg Zoletil (Virbac, TX, USA). To determine metabolites in the serum, the collected whole blood was centrifuged at 12,000 rpm using a Legend micro 17-R (Thermo Fisher Scientific, Waltham, MA, USA). TCHO-P III and GLU-P III levels were measured. Mice were euthanized using Zoletil over-dosing, then the abdominal fat tissues were collected and the weights of these tissues were measured.
8
Quantitative GUS Activity Assay
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The construction of gusA-based reporter strains for rpfF and rpfB was performed as previously described (28 (link)). Quantitative β-glucuronidase (GUS) activity assays were also performed as previously described (34 (link)). Briefly, the reporter strains were grown in XYS medium for 12 h to 36 h at 28°C. A sample of culture (1 mL) was then collected, centrifuged at 8,000 rpm (Thermo Scientific; Legend Micro 17R) for 10 min, and washed once with PBS buffer (1×, pH 7.4). Bacterial cells were then suspended in 1 mL PBS buffer following the addition of 20 μL of 0.1% (wt/vol) SDS solution and 40 μL of chloroform. Centrifugation was then conducted at 8,000 rpm for 5 min, and 100 μL of supernatant was removed and added to 250 μL of MUG solution (1 mM 4-methylumbelliferyl β-d -glucuronide, 50 mM PBS, 5 mM DTT, and 1 mM EDTA, pH 8.0). The reaction mixtures were subsequently incubated at 37°C for 15 min. Each reaction (200 μL) was terminated by mixing with 800 μL of 0.2 M Na2CO3 solution. A 96-well plate was used to detect GUS activities with a fluorescence microplate reader based on excitation and emission wavelengths of 365 nm and 455 nm, respectively.
9
Western Blot Assay for Protein Detection
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For the Western blot assay, the cell pellets were re-suspended in lysis buffer consisting of 50 mmol/L HEPES, pH 7.4; 1% Triton-X100; 2 mmol/L sodium orthovanadate; 100 mmol/L sodium fluoride; 1 mmol/L edetic acid; 1 mmol/L PMSF; 10 mg/L aprotinin (Sigma, MO, USA) and 10 mg/L leupeptin (Sigma) and were lysed at 4 °C for 1 h. After centrifugation at 12,000 rpm (Legend Micro 17R, Thermo Fisher Scientific, Waltham, USA) for 15 min, the protein content of the supernatant was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of total protein were separated by 10%–12% SDS-PAGE and were transferred to PVDF membranes, which were soaked in blocking buffer (5% skimmed milk or BSA). Proteins were detected by applying primary antibodies followed by HRP-conjugated secondary antibody and were visualized using ECL as the HRP substrate.
10
Quantitative Phytochemical Analysis of H. pluvialis
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The cell number density of H. pluvialis was determined using an improved Neubauer hemocytometer (DHC-N01-5; INCYTO, Chungnam, Korea). Morphological characteristics and cell diameter were analyzed using an Axiolab bright-field microscope equipped with a digital camera and Zen lite 2012 software (Carl Zeiss, Jena, Germany). Light intensity and pH were measured using an LI-250A quantum photometer (LI-COR Inc., Lincoln, NE, USA) and pH meter (HM-30R; TOADKK, Tokyo, Japan), respectively.
Chlorophyll concentration of H. pluvialis cultures was monitored based on solvent extraction and spectrophotometric assays according to a previous report [2] (link). Briefly, 1 mL of algal solution from the flask culture was collected in a bead-beating tube and centrifuged for 10 min at 10,000 rpm (Legend Micro 17R; Thermo Fisher Scientific, Waltham, MA, USA). After discarding the supernatant, 1.0 g micro-beads were added and resuspended in 1 mL acetone. The algal cells were mechanically disrupted using a bead beater (6 m/s for 30 s, 3 times). Chlorophyll a and b concentrations were estimated using the following equations (mg/L) based on the OD values at 644. 8 (link) Chlorophyll a and b = Chlorophyll a + Chlorophyll b
Chlorophyll concentration of H. pluvialis cultures was monitored based on solvent extraction and spectrophotometric assays according to a previous report [2] (link). Briefly, 1 mL of algal solution from the flask culture was collected in a bead-beating tube and centrifuged for 10 min at 10,000 rpm (Legend Micro 17R; Thermo Fisher Scientific, Waltham, MA, USA). After discarding the supernatant, 1.0 g micro-beads were added and resuspended in 1 mL acetone. The algal cells were mechanically disrupted using a bead beater (6 m/s for 30 s, 3 times). Chlorophyll a and b concentrations were estimated using the following equations (mg/L) based on the OD values at 644. 8 (link) Chlorophyll a and b = Chlorophyll a + Chlorophyll b
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