Myeloid immortalization assays using serial replating were performed as described [21 (link)]. Briefly, the purified CD34(-) or CD34(+) KSL (150) and MP (1500) cells were directly sorted into 1.5 ml of methylcellulose medium prepared from M3234 (StemCell Technologies, Vancouver, Canada) according to the manufacture’s protocol, supplemented with 25 ng/ml SCF, 10 ng/ml each of IL-6, mouse IL-3, mouse GM-CSF (Miltenyi Biotec), and 0.1 μM of 4-OHT. The sorted cells in 1 ml of the mixture were immediately plated in 35 mm dishes. Alternatively, retrovirally transduced KSL, CD34(+) KSL, and MP cells were plated in the same M3234-based medium supplemented with G418 in addition to the cytokines. After culturing for 5 (fo MP cells), 6 (for CD34(+) KSL cells), and 10 days (for retrovirally transduced cells and CD34(-) KSL cells), colonies were enumerated, and single-cell suspensions (0.3–1×104 cells) of colonies were subsequently replated in α-MEM-based medium containing 1% methylcellulose (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) supplemented with only the same cytokines. Every 5–7 days, replating of cells collected from colonies was repeated in the same way using the α-MEM-based methylcellulose medium. The immortalized cells were harvested from colonies in the third plating, and were cultured in α-MEM supplemented with 20% FBS and the same cytokines.
Methylcellulose
Manufactured by Shin-Etsu Chemical
Sourced in Japan
Methylcellulose is a water-soluble cellulose ether produced by Shin-Etsu Chemical. It is a white to off-white, odorless, and tasteless powder. Methylcellulose functions as a thickening, suspending, and emulsifying agent in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Sm 25, by Shin-Etsu Chemical (4 mentions)
M3234, by STEMCELL (1 mentions)
Mouse gm csf, by Miltenyi Biotec (1 mentions)
Mouse il 3, by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (1 mentions)
Precision brain slicer, by Braintree Scientific (1 mentions)
Fenofibrate, by Merck Group (1 mentions)
C57bl 6j mice, by CLEA Japan (1 mentions)
Pemafibrate, by Kowa (1 mentions)
Jnj7777120, by Merck Group (1 mentions)
2 hydroxypropyl β cyclodextrin, by Merck Group (1 mentions)
Mx 201, by Thinky (1 mentions)
Testosterone, by Tokyo Chemical Industry (1 mentions)
Astemizole, by Merck Group (1 mentions)
Verapamil hydrochloride, by Merck Group (1 mentions)
O desmethyl astemizole, by LGC (1 mentions)
Flecainide acetate, by Merck Group (1 mentions)
12 protocols using methylcellulose
1
Myeloid Immortalization Assays by Serial Replating
2
Edaravone Suspension Stability: Effect of Thickening Agents
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Example 4
200 mg of methylcellulose (Shin-Etsu Chemical Co., Ltd., SM-25) was dissolved in 200 mL of water to prepare a 0.1% (w/v) methylcellulose aqueous solution. 100 mg of edaravone particles (edaravone powder, D50: 37 μm; D90: 143 μm) was dispersed in 10 mL of the methylcellulose aqueous solution to obtain an edaravone suspension.
5 mL of each of the edaravone suspensions prepared in Examples 1-10 was put in a glass bottle and was sealed, and was stored at 60° C. for 4 weeks. After 4 weeks, an amount of edaravone-related substances in each suspension was measured according to Related Substances (i) in Purity Test described in Edaravone Injection in Japanese Pharmacopeia. The results are shown in the following table.
From the above results, it can be seen that, although an amount of edaravone-related substances generated in any one of Examples 1-10 is small, the amounts of the edaravone-related substances generated in the suspensions of Examples 1-6 in which xanthan gum or tragacanth powder was used as a thickening agent are particularly small.
3
Edaravone Suspension Stability Comparison
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Example 4
200 mg of methylcellulose (Shin-Etsu Chemical Co., Ltd., SM-25) was dissolved in 200 mL of water to prepare a 0.1% (w/v) methylcellulose aqueous solution. 100 mg of edaravone particles (edaravone powder, D50: 37 μm; D90: 143 μm) was dispersed in 10 mL of the methylcellulose aqueous solution to obtain an edaravone suspension.
5 mL of each of the edaravone suspensions prepared in Examples 1-10 was put in a glass bottle and was sealed, and was stored at 60° C. for 4 weeks. After 4 weeks, an amount of edaravone-related substances in each suspension was measured according to Related Substances (i) in Purity Test described in Edaravone Injection in Japanese Pharmacopeia. The results are shown in the following table.
From the above results, it can be seen that, although an amount of edaravone-related substances generated in any one of Examples 1-10 is small, the amounts of the edaravone-related substances generated in the suspensions of Examples 1-6 in which xanthan gum or tragacanth powder was used as a thickening agent are particularly small.
4
Edaravone Suspension Stability and Related Substances
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Example 4
200 mg of methylcellulose (Shin-Etsu Chemical Co., Ltd., SM-25) was dissolved in 200 mL of water to prepare a 0.1% (w/v) methylcellulose aqueous solution. 100 mg of edaravone particles (edaravone powder, D50: 37 μm; D90: 143 μm) was dispersed in 10 mL of the methylcellulose aqueous solution to obtain an edaravone suspension.
5 mL of each of the edaravone suspensions prepared in Examples 1-10 was put in a glass bottle and was sealed, and was stored at 60° C. for 4 weeks. After 4 weeks, an amount of edaravone-related substances in each suspension was measured according to Related Substances (i) in Purity Test described in Edaravone Injection in Japanese Pharmacopeia. The results are shown in the following table.
From the above results, it can be seen that, although an amount of edaravone-related substances generated in any one of Examples 1-10 is small, the amounts of the edaravone-related substances generated in the suspensions of Examples 1-6 in which xanthan gum or tragacanth powder was used as a thickening agent are particularly small.
5
IEGs Expression Analysis in Mouse Brain
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For IEGs expression analysis, ICR mice were used unless otherwise noted and habituated appropriately (as described above). Only for MAP and PCP dose-response experiments, C57BL/6J mice were used for experiments. Drugs were administered orally (p.o., suspended in 0.5% methylcellulose (Shin-Etsu Chemical)), intraperitoneally (i.p., dissolved in saline, sterile 0.9% NaCl (Otsuka Pharmaceutical Factory)) and subcutaneously (s.c., dissolved in saline same as the case of i.p.). At each time point, the mice were euthanized by decapitation and whole brains were collected. Fitting the brain on a Precision Brain Slicer (Braintree Scientific), four brain regions including prefrontal cortex, nucleus accumbens, striatum, and hippocampus were dissected on ice. Schematic representation of those brain regions is shown in S1 Fig . Here, prefrontal cortex was defined as the anterior part of the frontal lobe of the brain 1 mm behind the olfactory bulb. Nucleus accumbens, striatum, and hippocampus were dissected from both hemispheres with a 1 mm slice using a Precision Brain Slicer.
6
Edaravone Suspension Stability Evaluation
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Example 4
200 mg of methylcellulose (Shin-Etsu Chemical Co., Ltd., SM-25) was dissolved in 200 mL of water to prepare a 0.1% (w/v) methylcellulose aqueous solution. 100 mg of edaravone particles (edaravone powder, D50: 37 μm; D90: 143 pun) was dispersed in 10 mL of the methylcellulose aqueous solution to obtain an edaravone suspension.
5 mL of each of the edaravone suspensions prepared in Examples 1-10 was put in a glass bottle and was sealed, and was stored at 60° C. for 4 weeks. After 4 weeks, an amount of edaravone-related substances in each suspension was measured according to Related Substances (i) in Purity Test described in Edaravone Injection in Japanese Pharmacopeia. The results are shown in the following table.
From the above results, it can be seen that, although an amount of edaravone-related substances generated in any one of Examples 1-10 is small, the amounts of the edaravone-related substances generated in the suspensions of Examples 1-6 in which xanthan gum or tragacanth powder was used as a thickening agent are particularly small.
7
Oxygen-Induced Retinopathy Mouse Model
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C57BL/6J mice were obtained from CLEA Japan, Inc. OIR was induced in the mice by exposure to 85% oxygen from postnatal day 8 (P8) for 72 h. Pemafibrate (0.3 mg/kg/day; Kowa, Tokyo, Japan), fenofibrate (10 mg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) and vehicle (methyl cellulose: Shin-Etsu Chemical, Tokyo, Japan) were fed via oral gavage from P12 to P16 daily. Mice under normoxia with vehicle were also prepared (NOX group). At P17, the vaso-obliteration (VO) and neovascular tuft (NV) areas were evaluated on the wholemount retinae with iso-lectin B4 staining, as previously described [21 (link)].
8
Flutamide Administration in BSO Treatment
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The dose level of flutamide was set at 150 mg/kg. Flutamide was suspended in an aqueous solution of 0.5% methylcellulose (Shin-etsu Chemical Co., Ltd., Tokyo, Japan, 5 mL/kg). The administration of flutamide was started 2 days after the initiation of treatment with BSO (dosing duration of flutamide: 5 days). Necropsy was conducted on day 36 (Week 5).
9
Pharmacological Evaluation of Histamine Agents
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We purchased imetit dihydrobromide, ciproxifan maleate, and JNJ-7777120 from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Imetit was dissolved in saline and 0.5% methylcellulose (Shin-Etsu Chemical, Tokyo, Japan) solution for the electrophysiological tests and copulatory behavior tests, respectively. Ciproxifan was dissolved in saline for both electrophysiological and copulatory behavior studies. JNJ-7777120 was dissolved in 20% (2-hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, Inc., St. Louis, MO, USA) solution for the electrophysiological tests.
10
Formulation and Storage of KTZ Suspension
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Methylcellulose (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) was dissolved in injection-grade distilled water to make a 0.5% (w/v) solution. KTZ (Wako Pure Chemical Industries, Tokyo, Japan) was weighed and mixed with the solution using a defoaming conditioning mixer (MX-201, THINKY Corporation, Tokyo, Japan) to make a 0.5% (w/v) suspension of KTZ. Batches of the dosing suspensions sufficient for several days of dosing (maximum 5 days) were prepared and were stored in a refrigerator (set at 4°C) until use. Prior to dose administration, the dosing suspension was allowed to warm to room temperature. The dose volume for each animal was 5 Pimonidazole hydrochloride was purchased from HPI (Hypoxyprobe Plus kit, Burlington, MA), dissolved in physiological saline to give a 60 mg/mL solution and filter sterilized prior to intraperitoneal injection.
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