F. casuarinae was grown in liquid CB medium for approximately 7 d. Hyphae were collected by centrifugation (2,500×g, 20°C, 10 min) from a 14-mL culture and resuspended in 3 mL CB medium. Hyphae were homogenized by forced passages through a 21G needle (TERUMO, Tokyo, Japan). Fragmented hyphae were irradiated with GR (60Co) for 8 h (772 Gy) or 12 h (1158 Gy). After irradiation, hyphae were collected by centrifugation and resuspended in 1.1 mL BAP-T medium. Before and after mutagenesis, samples of the cell suspension were plated on solid CB media to assess survival rates. The cell suspension (0.1 mL) was inoculated into 10 mL CB liquid medium and the cultures were incubated at 28°C for a few weeks.
21 g needle
Manufactured by Terumo
Sourced in Japan
The 21G needle is a medical device used for various procedures. It has a gauge size of 21, which refers to the diameter of the needle. This needle is designed for various medical applications that require a specific gauge size.
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6 protocols using 21 g needle
1
Mutagenesis of the Fungus F. casuarinae
2
Dynamic Culture of Organoid Aggregates
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Dynamic culture of the organoids was conducted in a LiveBox bioreactor (LB; IVTec, Massarosa (LU), Italy). The bioreactor, composed of a transparent chamber, features flow inlet and outlet for perfusion with culture media. The system reproduces the typical volume of a 24-well plate. All the components were autoclaved before use. The content of aggregates from one AggrewellTM microplate was put in the LB bioreactor 48 h after seeding. In order to avoid loss of aggregates due to dynamic flow, they were resuspended in 1.5% alginate dissolved in 20 mM HEPES-NaOH at pH 7.4, filtered through a 0.45 μm filter, and autoclaved. The aggregates were collected from the AggrewellTM microplate, and the pellet was resuspended in 1 mL of alginate. To obtain the beads, the cell suspension was placed in a syringe provided with a 21G needle (Terumo, Tokyo, Japan), dropped in a solution of CaCl2 at 102 mM under magnetic stirring, and then left for 10 min at 4 °C. The aggregates were then cultured for 96 h at a flow rate of 330 mL/min, and half of the medium was changed 48 h after the starting point (Figure 1 ).
3
Porcine Oocyte Maturation Protocol
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Cumulus-oocyte complexes (COCs) were collected and used for IVM according to our previous method [31 (link)]. Briefly, ovaries
were collected from gilts at the slaughter house, stored at 37°C in phosphate-buffered saline containing antibiotics, and transported to the laboratory within 1
h. The COCs were aspirated from antral follicles (3–5 mm in diameter) using a syringe with a 21-G needle (Terumo, Tokyo, Japan). The COCs were cultured in IVM
medium for 44 h (10 COCs/100 μl drops) and then subjected to activation.
were collected from gilts at the slaughter house, stored at 37°C in phosphate-buffered saline containing antibiotics, and transported to the laboratory within 1
h. The COCs were aspirated from antral follicles (3–5 mm in diameter) using a syringe with a 21-G needle (Terumo, Tokyo, Japan). The COCs were cultured in IVM
medium for 44 h (10 COCs/100 μl drops) and then subjected to activation.
4
Porcine Oocyte Maturation Protocol
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The ovaries were collected from gilts at a slaughterhouse, stored at 37ºC in phosphate-buffered saline (PBS) containing antibiotics, and transported to the
laboratory within 1 h. Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3–5 mm in diameter) using a syringe with a 21 G needle (Terumo,
Tokyo, Japan). The COCs were cultured in IVM medium for 44 h (10 COCs/100 µl drops) and then subjected to activation.
laboratory within 1 h. Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3–5 mm in diameter) using a syringe with a 21 G needle (Terumo,
Tokyo, Japan). The COCs were cultured in IVM medium for 44 h (10 COCs/100 µl drops) and then subjected to activation.
5
Porcine Ovary Retrieval and COCs Isolation
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Porcine ovaries were collected from the slaughterhouse. Ovaries were transported to the laboratory in phosphate-buffered saline (PBS) containing antibiotics at 25℃ within 2 h. Cumulus cell and oocytes complexes (COCs) were collected from antral follicle (3-5mm in diameter) using a 21 G needle (Terumo, Tokyo, Japan) connected to a 10 ml syringe (Terumo).
This study was approved by the Ethics Committee for Animal Experiments of Tokyo University of Agriculture (2023009).
This study was approved by the Ethics Committee for Animal Experiments of Tokyo University of Agriculture (2023009).
6
Biotinylation and Extraction of Primary CD4+ T Cell Surface Proteins
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Primary human CD4 + T cells (5 x 10 6 cells) were washed three times in ice cold PBS (1mL; pH 8) and cell surface proteins labelled with 0.5 mg/mL biotin (Sulfo-NHS-SS-Biotin, Thermo Scientific, UK) for 20 minutes at 4 ºC on a rotary mixer. Cells were washed twice with glycine (10 mM) to quench the biotinylation reaction and washed once with PBS (pH 8). Cells were lysed on ice for 30 minutes in MNE (25mM MES (pH 6.5), 150mM NaCl, 2mM EDTA) lysis buffer supplemented with 1mM sodium orthovanadate, 1% v/v Triton X-100 and 0.1% v/v protease inhibitor cocktail (Sigma Aldrich, UK), genomic DNA was sheared using a 21 G needle (Terumo, UK) and centrifuged at 2,000 xg for 5 minutes to obtain a post-nuclear supernatant (PNS). The PNS (1mL) was incubated for 30 minutes at room temperature with 200 µL of pre washed Magnabind Streptavidin (SA) beads (Thermo Scientific, UK), an unbound fraction was collected and beads were washed twice in MNE lysis buffer and twice in PBS (pH 8). Cell surface proteins were eluted into extraction buffer (8M urea, 2M thiourea, 2% w/v CHAPS, 1% v/v DeStreak reagent (GE Healthcare, Amersham, UK) and 0.2% v/v Bio-Lytes (Bio-Rad)) for 30 minutes at room temperature. PNS, unbound and elution fractions were stored at -80 ºC for SDS-PAGE analysis and western blotting. The yield of CD4+ T cell membrane proteins eluted ranged from 8-20µg for the 19 subjects studied.
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