After the 50‐L cell culture (day 27), 300‐mL samples (approximately 0.34 g microcarriers) for cell harvest were taken from the bioreactor under agitation. Once the microcarriers settled, the supernatant medium was removed and PBS(−) was used to wash the microcarriers twice. Twenty milliliters of TrypLE Select enzyme solution (Thermo Fisher Scientific) was added to the microcarriers, which was put on a bioshaker (Taitec) and oscillated at 100 strokes per minute for 12 minutes at 37°C. Detachment of the cells from the microcarriers were confirmed under a phase contrast microscope (OLYMPUS). Culture medium (30 mL) was added to the suspension of detached cells and microcarriers, which was passed through a Falcon mesh (hole size: 50 μm; Corning) to separate the cells from microcarriers. The harvested cells were suspended at 5 × 106 cells/mL in STEM‐CELLBANKER GMP grade (ZENOAQ RESOURCE Co, Ltd), aliquoted in 1.5‐mL cryotubes (Thermo Fisher Scientific) and stored in BICELL freezing vessels (Nihon Freezer Co, Ltd) at −80°C for 24 hours. Subsequently, the frozen cells were transferred in a liquid nitrogen tank and used for characterization.
Bioshaker
Manufactured by TAITEC
Sourced in Japan
The Bioshaker is a laboratory equipment designed for mixing and agitating samples. It provides a consistent and controlled motion for efficient sample preparation and incubation.
Automatically generated - may contain errors
Lab products found in correlation
Cell strainer, by BD (2 mentions)
Liberase tl research grade, by Roche (2 mentions)
Dnase, by Fujifilm (2 mentions)
Phase contrast microscope, by Olympus (1 mentions)
Cryotube, by Thermo Fisher Scientific (1 mentions)
Tryple select enzyme solution, by Thermo Fisher Scientific (1 mentions)
Du730, by Beckman Coulter (1 mentions)
Haucl3 3h2o, by Merck Group (1 mentions)
Multisizer 4 coulter counter, by Beckman Coulter (1 mentions)
Coulter isoton 2 diluent, by Beckman Coulter (1 mentions)
8 protocols using bioshaker
1
Microcarrier-based Cell Harvest and Cryopreservation
2
Protein Adsorption on Nanoparticles
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A solution containing 40 µg of protein was added to 2.5 mg of inorganic oxide nanoparticles in 1.5 mL microtubes, and the suspended mixture was briefly sonicated to disperse inorganic oxide nanoparticles in solution. The mixture was then shaken in a bioshaker (Taitec, Saitama, Japan) overnight at room temperature for protein adsorption on the particles. The supernatants, defined as the unadsorbed fraction, were then separated by centrifugation at 10,000× g for 10 min. Finally, the protein contents in the unadsorbed fractions were quantified by the BCA method.
3
Monitoring Bacterial Growth Dynamics
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A total of 43 mL of M63 medium was placed in a 50 mL Falcon (Watson) tube in which 100 μL of the E. coli glycerol stock stored at − 80 °C was added and well mixed using a vortex. A total of 5 mL of the mixture was dispensed into eight sterilized glass tubes and cultured at 37 °C and 200 rpm using a bioshaker (Taitec). The culture was temporally sampled from the late lag phase to the early stationary phase to detect the growing population using three different methods that relied on the physical, biological and chemical properties of bacterial cells, i.e., the optical turbidity, colony formation, and ATP abundance. Sampling was conducted by taking 1.2 mL of the culture from two out of eight glass tubes, and 1 mL of the sampled culture was placed in a cuvette to measure absorbance at OD600 (Beckman DU730), and the remaining volume was used for the other two assays. Time sampling was performed rotationally among a total of eight glass tubes, which led to a single growth curve based on the OD600 value. This time course of bacterial growth was conducted repeatedly (N = 3~ 5). The biological replications varied from the initial pre-culture.
4
Isolation of Epidermal and Dermal Cells
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Epidermal and dermal cell suspensions were prepared as before4 (link) with slight modification. Briefly, shaved whole trunk skin was harvested from appropriate animals and, after removal of subcutaneous tissues with forceps, was floated with epidermal side up onto 10 mL of Trypsin-EDTA solution containing 5 mL of 0.25% Trypsin (Nacalai tesque) and 5 mL of 0.05% Trypsin-0.53 mM EDTA • 4Na (Nacalai tesque) at 37°C for less than 30 minutes in 10 cm dish. Epidermis and dermis were separated manually with forceps. Dermis was cut into small chips manually with scissors and further incubated in 4 mL of RPMI containing 0.03% Liberase TL Research grade (Roche Applied Science) and 200 U/mL of DNase (Wako Pure Chemical Industries) for 60 minutes at 37°C shaking with rotation at 200 rpm (Bioshaker, Taitec). Epidermal and dermal cells were then suspended in 5% FCS in PBS, washed and filtered through Cell strainer (BD Falcon).
5
Isolation of Epidermal and Dermal Cells
6
Lipid Micelle-Liposome Fusion Protocol
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6 mol% RGD-(SG)5-lipid micelles or 9 mol% cRGD-(SG)5-lipid micelles were incubated with PEGylated liposomes while shaking at 150 rpm and 60 °C on a bioshaker (Taitec, Saitama, Japan) for 1 h or 1 min. Each liposome sample was dispersed in 1× phosphate-buffered saline (PBS) (pH 7.4) by adding 10× PBS and then filtered using a 0.45-μm filter.
7
Chaga Extract-Mediated Gold Nanoparticle Synthesis
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The Ch-AuNPs synthesis involved the reaction of 5 mL Chaga extract with different ratios of 0.001 M aqueous solution of HAuCl3·3H2O (Sigma, Shanghai, China). The reactions were performed in a shaking incubator (BioShaker, Taitec, Sausal, Austria) at 150 rpm set at 30–70 °C (Table 3 ) for 24 h.
Two different concentration ratios of extract and gold chloride solution, 1:1 and 1:2, respectively, were selected for this purpose. The solutions, post reaction, were centrifuged at 15,000 rpm for 30 min at room temperature to collect the Ch-AuNPs. The Ch-AuNPs were further resuspended in Milli-Q water and vortexed for approximately 1 min and centrifuged again at 15,000 rpm for 10 min. This process was repeated 3 times to remove the excess unreacted components of both the extract and the HAuCl3·3H2O. The final washed Ch-AuNPs were dispersed in Milli-Q water and stored at room temperature for further use. The synthesis process is demonstrated inFigure S17 .
Two different concentration ratios of extract and gold chloride solution, 1:1 and 1:2, respectively, were selected for this purpose. The solutions, post reaction, were centrifuged at 15,000 rpm for 30 min at room temperature to collect the Ch-AuNPs. The Ch-AuNPs were further resuspended in Milli-Q water and vortexed for approximately 1 min and centrifuged again at 15,000 rpm for 10 min. This process was repeated 3 times to remove the excess unreacted components of both the extract and the HAuCl3·3H2O. The final washed Ch-AuNPs were dispersed in Milli-Q water and stored at room temperature for further use. The synthesis process is demonstrated in
8
Measuring E. coli Cell Size Dynamics
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The E. coli culture stocks were inoculated in 3 ml of M63 minimal medium at a 100-fold dilution and were cultured in a bioshaker (Taitec) with a rotation rate of 200 rpm at 37 °C. Each cell culture was temporally sampled for size measurement from the late exponential to stationary phases. The cell size (diameter) and the corresponding cell concentration were measured using a cell counter (Multisizer™ 4 coulter counter, Beckman) equipped with a 20-μm aperture. Every 50 μl of cell culture, a 5- to 10-fold dilution, if required, was mixed with 10 ml of COULTER ISOTON II DILUENT (Beckman) in a 25-ml disposable vial (Accuvette ST, Beckman) for measurement. The distribution of cell sizes, including the mean, medium and standard deviation of the distribution, were acquired automatically at each measurement (30,000∼100,000 cells). Repeated experiments (from cell culture to size measurement) were performed for each strain. The average of multiple values of the measured medium was taken to be the representative cell size of the cell population. The stationary cell sizes were determined as the cell size acquired at concentrations >1 × 109 cells/ml, individually. Six to eight measurements during stationary phase were obtained for each strain, and the average of these measurements was applied to the analysis.
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