DO11.10 T hybridoma, 2B4.11 T hybridoma, IIA1.6, and CH27 cells were maintained in RPMI 1640 medium (Gibco), supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest), 0.5 mM Monothioglycerol (Wako), 2 mM L-alanyl-L-glutamine dipeptide (Gibco), 100 U/ml penicillin (Nacalai Tesque), and 100 μg/ml streptomycin (Nacalai Tesque). Plat-E cells were maintained in Dulbecco's Modified Eagle Medium (D'MEM, Invitrogen), supplemented with 10% (v/v) FBS, 100 U/ml penicillin (Nacalai Tesque), and 100 μg/ml streptomycin (Nacalai Tesque).
Monothioglycerol
Manufactured by Fujifilm
Sourced in Japan, Germany, United States
Monothioglycerol is a chemical compound used in various laboratory applications. It functions as a reducing agent and is commonly employed in analytical procedures, biochemical assays, and the formulation of certain reagents. The core purpose of Monothioglycerol is to provide a controlled reducing environment for specific experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Bovine insulin, by Merck Group (3 mentions)
L alanyl l glutamine, by Fujifilm (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
L alanyl l glutamine dipeptide, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
B27 supplement minus vitamin a, by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Chicken serum, by Thermo Fisher Scientific (1 mentions)
Embryomax nucleosides, by Merck Group (1 mentions)
Human fgf2, by Thermo Fisher Scientific (1 mentions)
Blebbistatin, by Fujifilm (1 mentions)
Antibiotic antimycotic mixed stock solution, by Nacalai Tesque (1 mentions)
12 protocols using monothioglycerol
1
Cell culture protocol for T-cell hybridomas
2
Maintenance of IPKM and PAM cells
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IPKM cells were routinely maintained as described previously [10 (link),11 (link)]. Briefly, the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 10 µg/mL bovine insulin (Merck, Darmstadt, Germany), 25 µM monothioglycerol (Wako, Osaka, Japan), and antibiotics in cell culture plates and flasks (Sumitomo Bakelite, Tokyo, Japan). Porcine alveolar macrophage (PAM) cells were prepared from 8-week-old LWD pigs as described previously [10 (link)] and stored at −80 °C until use.
3
Avian Primordial Germ Cell Culture
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Whole blood samples containing PGCs were collected from a BPR embryo at Hamburger Hamilton (HH) stage 13–15 (Hamburger and Hamilton, 1951 ). The blood was dispersed in 500 µL of PGC culture medium. For each culture experiment, PGCs derived from a single embryo were used. The PGC culture medium used was same as that described in a previous study (Ezaki et al., 2020 ) with some modifications. Briefly, KnockOut DMEM (Thermo Fisher Scientific, Waltham, MA, USA) was supplemented with 1X B-27 Supplement Minus Vitamin A (Thermo Fisher Scientific), 1% Chicken Serum (Thermo Fisher Scientific), 1X EmbryoMAX nucleosides (Merck, Darmstadt, Germany), 1X MEM non-essential amino acids (Thermo Fisher Scientific), 0.5 mM monothioglycerol (Wako Pure Chemical Industries, Osaka, Japan), 1X Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque, Kyoto, Japan), 10 ng/mL human FGF2 (PeproTech, Rocky Hill, NJ, USA), 1 unit/mL heparin (Merck), 0.2 µM blebbistatin, and 0.2 µM H-1152 (Wako Pure Chemical Industries). Whole blood samples containing PGCs were cultured in 24-well plates without feeder cells at 38°C, 5% CO2, and 3% O2, and subcultured every 2–4 days. The PGCs were passaged based on their growth. The PGCs were frozen in STEM-CELLBANKER (Nippon Zenyaku Kogyo, Fukushima, Japan) and stored at −80°C.
4
Porcine Kidney Macrophage Cell Line for ASFV
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IPKM was established by immortalizing porcine primary culture of kidney macrophages with recombinant lentivirus vectors as previously described [20 (link)]. IPKM is highly susceptible to field ASFV isolates and cell-adapted ASFV isolates [21 (link),22 (link)]. The cells were routinely maintained in growth medium (Dulbecco’s modified Eagle’s medium (Nakali Tesque, Kyoto, Japan)) supplemented with 10% fetal bovine serum (FBS), 10 μg/mL bovine insulin (Merck, Darmstadt, Germany), 25 μM monothioglycerol (Wako, Osaka, Japan)), and antibiotics in cell culture plates and flasks for suspension culture (Sumitomo Bakelite, Tokyo, Japan). Porcine alveolar macrophage (PAM) cells were prepared from 8-week-old Landrace (L), Yorkshire (W), and Duroc (D) cross breed (LWD) pigs as described previously and stored at −80 °C.
5
Ocular Cell Differentiation from iPSCs
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The differentiation of ocular cells from human iPSCs was performed according to the previously reported method14 (link). Briefly, human iPSCs were seeded onto LN511E8-coated (0.5 μg/cm2) plates and cultivated in StemFit medium for 10 days, after which the medium was changed to serum-free differentiation medium [DM; GMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% knockout serum replacement (KSR; Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM non-essential amino acids (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% Penicillin-Streptomycin solution (Life Technologies) and 55 μM 2-mercaptoethanol (Life Technologies) or monothioglycerol (Wako, Osaka, Japan)]13 (link),14 (link). After 4 weeks, the medium was replaced with corneal differentiation medium (CDM) supplemented with 10–20 ng/mL KGF (Wako) and 10 μM Y-27632 (Wako) and cultured for an additional 4 weeks. Non-epithelial cells were removed from the SEAM by pipetting at around week 7 after initiation of differentiation. Following this, the medium was replaced with corneal epithelium maintenance medium (CEM) containing 2% B27 supplement (Life Technologies), 10–20 ng/mL KGF, and 10 μM Y-27632 at week 8, and the plates were incubated for an additional 2 to 7 weeks (i.e. 10–15 weeks in total).
6
Directed Differentiation of T Cells
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Clumps of T-iPSC were transferred onto C3H10T1/2 feeder cells and cultured in Sac medium supplemented with 20 ng/ml BMP-4 (R&D) under hypoxia (5% O2) for 6 days. Sac medium consists of IMDM (Sigma-Aldrich) supplemented with 15% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, 1% insulin–transferrin–selenium, 450 mM monothioglycerol (Nacalai), 50 µg/ml l -ascorbic acid 2-phosphate, and 50 ng/ml VEGF (Wako). The culture condition was returned to normoxia after 6 days hypoxic culture and changed to Sac medium supplemented with 30 ng/ml SCF (R&D) and 10 ng/ml Flt3L (Peprotech). On day 14, HPC were collected and transferred onto OP9DL1 cells in OP9 medium supplemented with 1% insulin–transferrin–selenium, 50 µg/ml l -ascorbic acid 2-phosphate, 1 ng/ml IL-7, and 10 ng/ml Flt3L. After 3 weeks culture on OP9DL1, DP cells underwent the maturation process.
7
Lacrimal Gland Differentiation from hiPS Cells
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Multi zonal ocular cell differentiation approach developed by Li and others was adopted for lacrimal gland differentiation (Li et al., 2019 (link)). hiPS cells were seeded on 1% hESC qualified Matrigel-coated six well plates and maintained in eye field differentiation (ED) medium composed of (DMEM/F12 (Gibco, Thermo Fisher Scientific) and neurobasal medium (Gibco, Thermo Fisher Scientific) (1:1) supplemented with 2 mM L-GlutaMAX (Gibco, Thermo Fisher Scientific), 0.1 mM non-essential amino acids (Lonza), 0.1 mM monothioglycerol (FUJIFILM Wako Pure Chemical Corporation), and 1% N2 MAX supplement (R&D Systems). 2% Matrigel was added to ED medium for first 2 days of differentiation. After 7 days of culture, medium was replaced with the ocular cell differentiation (OD) medium composed of DMEM/F12 supplemented with 10% knockout serum replacement (Gibco, Thermo Fisher Scientific), 2 mM L-GlutaMAX, 0.1 mM NEAA, and 0.1 mM monothioglycerol. Organoids maintained in culture for up to 45 days with daily medium refreshments.
8
Maintenance of iPSCs and iPSECs
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iPSCs were maintained on MMC-treated MEFs in iPS medium containing DMEM/F12 (Wako) supplemented with 20% KnockOut Serum Replacement (Invitrogen), 2 mmol/L l -Alanyl-l -Glutamine (Wako), 0.1 mmol/L monothioglycerol (Wako), 0.5% penicillin and streptomycin (Nacalai Tesque, Kyoto, Japan) and 5 ng/ml basic fibroblast growth factor (Wako). The iPSECs were maintained on collagen I-coated dishes with HuMedia-EB2 medium (KURABO, Japan), supplemented with 20 ng/ml VEGF (R & D Systems), 25 ng/ml bFGF, 0.5% penicillin and streptomycin, and 10% fetal bovine serum (FBS).
9
Culturing INS1E and Rat Pancreatic Fibroblasts
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The INS1E cell line, a gift from Prof. Pierre Maechler,14 (link) was maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco), 0.5 mM monothioglycerol (Antioxidant, Wako, Osaka, Japan), 2 mM L -alanyl-L -glutamine (Wako), and 50 μg/ml gentamicin sulfate (Wako). Rat pancreatic fibroblasts were cultured under the same conditions as INS1E cells. Rat neonatal pancreases were collected and enzymatically digested in a collagenase/proteinase cocktail (0.1% collagenase L, Nitta Gelatin, Osaka, Japan, 0.2% dispase II, Gibco in HBSS, Gibco) for 60 mins in a reciprocating water bath shaker at 37°C. Undigested debris was removed using a nylon mesh and the cells were cultured as described below. Attached fibroblasts proliferated continuously and were sequentially passaged (sub-cultured in another dish), effectively diluting any contaminating islets and blood cells. Pancreatic fibroblasts exhibited cellular senescence after 3 to 6 passages, and were used before cell division had slowed down.
10
Insulin Secretion Assay in INS-1E Cells
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The rat insulinoma cell line INS-1E was grown in advanced RPMI-1640 medium (Thermo Fisher
Scientific Inc., Waltham, MA, USA) containing 5% FBS, 0.5 mM monothioglycerol (FUJIFILM
Wako Pure Chemical Corp.) and 2 mM L-alanyl-L-glutamine (FUJIFILM Wako Pure Chemical
Corp.) [18 (link)]. Cells were seeded in 24-well plates (1
× 105 cells per well) and cultured for 2 days at 37°C in 5% CO2. The
medium was removed and INS-1E cells were preincubated for 1 h (5% CO2, 37°C) in
RPMI-1640 (glucose free; FUJIFILM Wako Pure Chemical Corp.) with 5% FBS and 100 mg/dl
glucose. Next, the cells were incubated for 1 h in a fresh batch of the same composition
medium with added 1,5-AG or TI. To determine the effects of 1,5-AG and TI on insulin
secretion, INS-1E cells were incubated in RPMI-1640 containing various concentrations of
1,5-AG (0, 10, 100 and 1,000 µM) and TI (0, 1, 10 and 100
µg/ml). Samples were collected in Protein LoBind® tubes and
insulin in the medium was determined with a Rat Insulin ELISA Kit (Morinaga Institute of
Biological Science, Inc.) according to the manufacturer’s protocol. To each well,
200-µl of M-PERTM Mammalian Protein Extraction Reagent
(Thermo Fisher Scientific Inc.) was added to dissolve cells for the determination of
cellular protein content with a PierceTM BCA Protein Assay Kit (Thermo Fisher
Scientific Inc.).
Scientific Inc., Waltham, MA, USA) containing 5% FBS, 0.5 mM monothioglycerol (FUJIFILM
Wako Pure Chemical Corp.) and 2 mM L-alanyl-L-glutamine (FUJIFILM Wako Pure Chemical
Corp.) [18 (link)]. Cells were seeded in 24-well plates (1
× 105 cells per well) and cultured for 2 days at 37°C in 5% CO2. The
medium was removed and INS-1E cells were preincubated for 1 h (5% CO2, 37°C) in
RPMI-1640 (glucose free; FUJIFILM Wako Pure Chemical Corp.) with 5% FBS and 100 mg/dl
glucose. Next, the cells were incubated for 1 h in a fresh batch of the same composition
medium with added 1,5-AG or TI. To determine the effects of 1,5-AG and TI on insulin
secretion, INS-1E cells were incubated in RPMI-1640 containing various concentrations of
1,5-AG (0, 10, 100 and 1,000 µM) and TI (0, 1, 10 and 100
µg/ml). Samples were collected in Protein LoBind® tubes and
insulin in the medium was determined with a Rat Insulin ELISA Kit (Morinaga Institute of
Biological Science, Inc.) according to the manufacturer’s protocol. To each well,
200-µl of M-PERTM Mammalian Protein Extraction Reagent
(Thermo Fisher Scientific Inc.) was added to dissolve cells for the determination of
cellular protein content with a PierceTM BCA Protein Assay Kit (Thermo Fisher
Scientific Inc.).
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