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34 protocols using filter paper

1

Polysaccharide-Enriched Extraction from Polygonum multiflorum

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PMR powder (10.0 g), grounded from roots of Polygonum multiflorum Thunb., was placed in a 250-ml round-bottomed flask and dissolved in 100 ml 90% ethanol or distilled water. The solutions were refluxed for 1 h before being filtered through a paper filter (Advantec, Tokyo, Japan). These were then evaporated to dryness with a rotary evaporator to yield final ethanol (PMREtOH) and water (PMRwater) extracts of 2.05 g and 4.16 g, respectively. To fractionate the PMRwater, 0.64 g of this extract was dissolved in 10 ml water and then added to 40 ml EtOH. The mixture was stored at 4 °C for 16 h, then centrifuged at 7,000 rpm for 15 min and filtered through a paper filter (Advantec). The precipitate was washed twice with 20 ml water and dried to yield a polysaccharide-enriched fraction (PSenrich) of ∼ 32 g, whereas the filtrate was evaporated to dryness with a rotary evaporator to yield a polysaccharide-depleted fraction (PSdeplete) of ∼ 0.23 g.
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2

Survival Assay of RNAi-Treated Adult Male Insects

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Adult males were injected with NcSP75 or EGFP dsRNAs within 24 h after eclosion. To examine the survival rate, 3–5 males were confined in a small plastic dish containing an artificial diet that was covered with two layers of stretched-parafilm M (Bemis, WI, USA). The artificial diet consisted of 5% sucrose and 0.005% riboflavin in distilled water, which was filtered through a 0.22-μm syringe filter (Millipore, MA, USA). A filter paper (Advantec, Tokyo, Japan) was placed on the bottom of the dish to absorb the honeydew droplets, thereby preventing insects from sticking to the dish. The artificial diet was changed every 2–3 days. The number of surviving individuals was recorded every day (NcSP75, n = 67 and EGFP, n = 66). The numbers are calculated from two independent experiments (S3 Fig).
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3

Tail Bleeding Time in Rats

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The rats were anaesthetised with thiopental sodium (75 mg/kg, i.p.). After blinding of the experimenter, an incision was made (depth 1 mm) with a blade (No. 11, FEATHER Safety Razor Co., Ltd.) on the artery of the ventral part of the tail at 4 cm from the tip, and the blood was blotted every 15 s with filter paper (No. 2, Advantec Toyo Kaisha, Ltd.) for 30 min. The bleeding time was defined as the multiplication of the number of detectable blood stains on the opposite side of the filter paper that touched the blood by 15 s.
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4

Affinity Purification of Recombinant Abaecin

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The E. coli BL21 transformed with 6xHisSUMO-abaecin:pKSEC1 were grown and treated as described above. After sonication, the supernatant was filtered through filter paper (Advantec, Japan) and 0.45 μm syringe filter (Minisart syringe filter, Sartorius stedim biotech, Germany). The filtrate was incubated with His 60 Ni Suferflow resin (Takara, Japan) by inverting slowly for 1 h at 4 °C. After the binding incubation, the column was washed with 50 column volumes of wash buffer (20 mM Tris, 300 mM sodium chloride, 50 mM Imidazol, pH 8.0). 6xHisSUMO-abaecin fusion proteins bound to Ni resins were eluted approximately with 5 column volumes of elution buffer (20 mM Tris, 300 mM sodium chloride, 250 mM Imidazol, pH 8.0). Recombinant abaecin was isolated from 6xHisSUMO by treatment of SUMO protease (Enzynomics, South Korea) at 30 °C for 6 h. To confirm the cleavage, SDS-PAGE was performed using NuPAGE™ 4–12% Bis-Tris Gel (Invitrogen, USA) with MES buffer.
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5

Preparation of P. ginseng Extract

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The P. ginseng dried roots (400 grams) were soaked in 70% ethanol (3 liters) at room temperature for 1 day, extracted three times for 1 hour with 70% ethanol in an ultrasonic apparatus, and lyophilized by using a freeze dryer (Operon, Seoul, Korea) to produce a 70% ethanol crude extract, which was filtered through filter paper (Advantec, Toyo Roshi Kaisha, Japan). The ethanol extract was evaporated under reduced pressure by using a rotary evaporator (R-205, Büchi, Germany) and lyophilized with freeze dryer (Operon, Seoul, Korea) to produce a 70% ethanol crude extract (80 grams, yield 20%).
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6

Standardized Korean Herbal Extract Preparation

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Standardized 5-yr-old Korean WG and RG were purchased from Gwangmyung Natural Pharmaceutical Co. (Busan, Korea); voucher specimens (No. 201KWG and 201KRG) were deposited at the Herbarium of the School of Korean Medicine, Pusan National University. WG and RG (1 kg) were finely ground and extracted with 10 times their volumes of 80% methanol at room temperature for 3 d and then the extraction process was repeated three times. After filtration using filter paper (Advantec, Tokyo, Japan), methanol was removed using a vacuum evaporator (Eyela, Tokyo, Japan) at 45°C, and the resulting extracts (WG and RG) were stored at −20°C until required. OVA (Grade V) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Before use, OVA was detoxified using a DetoxiGel column (Pierce, Rockford, IL, USA).
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7

Termite Collection and Rearing for Analysis

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One colony of C. gestroi and four colonies of G. sulphureus were collected in Universiti Sains Malaysia (USM) campus and Teluk Bahang, Pulau Pinang. Termites were brought to the Household and Structural Urban Entomology Laboratory, School of Biological Sciences, USM for further analysis. They were placed in a plastic container (covered with black plastic) supplied with soil and wood as food sources and were kept in the dark. They were reared at room temperature (28 ± 2°C) with a relative humidity of 70% ± 10). Prior to the experiment, termites were previously separated from debris using the bridging method by allowing them to access five stacks of pre-wetted pine blocks (20 × 10 cm) (Tamashiro et al. 1973 (link)). Then, the termites were counted and transferred to a plastic Petri dish (90 mm × 15 mm, Ideal Healthcare, Malaysia) lined with moistened filter paper (90 mm, Advantec, Japan).
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8

Rice 'Nuruk' Extraction and Characterization

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First, 20 g of rice 'Nuruk' was placed in a 200 mL Erlenmeyer flask, to which 100 mL of distilled water was then added. The flask was sealed with plastic wrap and at room temperature (25℃) for 3 h with occasional mixing for extraction. The sample was then filtered using filter paper (No.2, Advantec Co., Ltd., Tokyo, Japan), and the filtrate was used for the following experiment. The pH of the filtrate was measured using a pH meter (Orion 3 Star Benchtop pH meter, Thermo Fisher Scientific Inc., Waltham, MA, USA), and the acidity was measured based on the volume (in mL) of 0.1 N NaOH used to titrate 10 mL of the filtrate until the color of the sample changed to light green (Editors of Brewing Society of Japan, 1993). To measure the amino acidity, 10 mL of the filtrate was added to a flask and titrated against 0.1 N NaOH until the color changed to light pink. Next, 5 mL of a neutral formalin solution was added to restore the original color of the sample. Again, 0.1 N NaOH was added until the light pink color appeared, and the total amount of titrating solution used was determined. The soluble solid content (°Brix) was measured using a digital refractometer (PR-201, Atago Co., Tokyo, Japan). The reducing sugar content was measured using a modified DNS method.
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9

Microbial Biomass Carbon Analysis

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For microbial biomass C (MBC) analysis, subsamples from each of the six microcosms were randomly paired (e.g., jars 1&3, 2&4, and 5&6) and combined to form triplicate samples. MBC was measured following a standard fumigation-extraction method72 (link). Briefly, one subsample (1.33 g dry wt. equivalent) was immediately extracted by agitating for one hour in K2SO4 (0.05 M, 10 mL), while the other subsample was fumigated for 24 hours with ethanol-free chloroform (0.4 mL) prior to extraction with K2SO4. The extracts were passed through filter paper (0.45 μm, Advantec) and dissolved organic C was measured by combustion catalytic oxidation (TOC-VCPN TOC analyzer Shimadzu, Japan). A portion of the extract was freeze-dried, and total C and δ13C of the freeze-dried residue were measured using isotope ratio mass spectrometry (Delta V, Thermo Scientific, Germany) coupled to an elemental analyzer (NC2500, Carlo Erba, Italy).
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10

Dopamine Receptor Antagonist Effects on Termite Larva

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Just after the No. 1 larva emerged, reproductives (queen and king) and the No. 1 larva were transferred to a 46 mm Petri dish containing a moistened filter paper (Advantec, Tokyo, Japan). After 24 h (day 1 after the appearance of the No. 1 larva), the No. 1 larvae were placed on ice for 90 s to reduce movement, and an equal volume (32.2 nl) of double-distilled water (DDW) as a control or DA receptor antagonist, cis-(Z)-flupenthixol dihydrochloride (MP Biomedical, Santa Ana, CA, USA), diluted with DDW (100 pM and 1 nM) was injected using Nanoliter 2000 (World Precision Instruments, Sarasota, FL, USA) using a glass capillary (10–30 μm diameter) (DDW (n=9), DA antagonist 100 pM (n=10) and 1 nM (n=10)). Each solution was coloured with Fast Green FCF (Tokyo Chemical Industry, Tokyo, Japan), and injection within the body was confirmed. Each dish with the solution-injected No. 1 larva and reproductives was maintained in constant darkness at 25°C. On the lid of each dish, a cross was drawn for the observation of locomotor activity (see below). The female reproductive (queen) was marked on her abdomen with waterproof ink.
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