Stage 7 medium was based on a previous report [7 (link)] with our original modifications. Cells were cultured in the MCDB 131 medium with 1% P/S, 2% fat-free bovine serum albumin (FUJIFILM Wako), 20 mM glucose, 1.5 g/L NaHCO3, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5iII or the alternative candidates, 1 μM T3, 10 μM ZnSO4 (Merck Millipore), 1.4 IU/mL heparin sodium salt (Nacalai Tesque, Kyoto, Japan), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck Chemicals, Houston, TX, USA), 1 μM PD-166866, 3 μM TR06141363 (Multi-kinase inhibitor, Takeda Pharmaceutical Company), and 10 μM Y-27632, for 4 days. To generate iPICs for implantation, cells were dissociated and re-sized in an Elplasia 6-well microwell plate (Corning Incorporated, Corning, NY, USA) or a gas-permeable microwell culture bag (Toyo Seikan Group Holdings, Ltd., Yokohama, Japan) [20 (link)] and cultured in the stage 7 medium.
Heparin sodium salt
Manufactured by Nacalai Tesque
Sourced in Japan
Heparin sodium salt is a white or almost white crystalline or amorphous powder. It is a naturally occurring anticoagulant substance that is extracted from animal tissues, typically from intestinal mucosa of mammals.
Automatically generated - may contain errors
Lab products found in correlation
Theophylline, by Fujifilm (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Tcm 199 medium, by Thermo Fisher Scientific (3 mentions)
Trolox, by Fujifilm (2 mentions)
Its x, by Thermo Fisher Scientific (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Znso4, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Hyaluronan sodium salt, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Chondroitin sulfate c sodium salt, by Fujifilm (1 mentions)
Micromanipulator, by Narishige (1 mentions)
10 protocols using heparin sodium salt
1
Directed Differentiation of iPICs for Implantation
2
Thermosensitive Suicide Vector Synthesis
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Hyaluronan sodium salt was purchased from Sigma Aldrich. Sodium salts of chondroitin sulfates A and C were obtained from Wako Pure Chemical Industries, and heparin sodium salt from Nacalai Tesque. A thermosensitive suicide vector, pSET4s, was kindly provided by Dr. Takamatsu (National Agriculture and Food Research Organization).
3
Streptobacillus moniliformis Genome and Expression
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Streptobacillus moniliformis DSM 12112 and its genome were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen. Its complete genome sequence is available in GenBank under accession number, CP001779. This bacterium was statically cultured at 37 °C under 5% CO2 in 0.8% nutrient broth (0.3% beef extract and 0.5% peptone) (Difco) containing 20% horse serum (Thermo Fisher Scientific) for 24–48 h. To investigate expression of Smon0127 and Smon0123, S. moniliformis cells were cultured in nutrient broth and horse serum in the presence or absence of 0.2% GAG [hyaluronic acid (Fluka), chondroitin sulfate C sodium salt (Wako), or heparin sodium salt (Nacalai tesque)] dialyzed against pure water. Detailed experimental procedures for construction of the overexpression system of S. moniliformis proteins and protein purification are presented in Supplementary Information.
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Streptobacillus moniliformis DSM 12112 and its genome were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen. Its complete genome sequence is available in GenBank under accession number, CP001779. This bacterium was statically cultured at 37 °C under 5% CO2 in 0.8% nutrient broth (0.3% beef extract and 0.5% peptone) (Difco) containing 20% horse serum (Thermo Fisher Scientific) for 24–48 h. To investigate expression of Smon0127 and Smon0123, S. moniliformis cells were cultured in nutrient broth and horse serum in the presence or absence of 0.2% GAG [hyaluronic acid (Fluka), chondroitin sulfate C sodium salt (Wako), or heparin sodium salt (Nacalai tesque)] dialyzed against pure water. Detailed experimental procedures for construction of the overexpression system of S. moniliformis proteins and protein purification are presented in Supplementary Information.
4
Bovine Oocyte In Vitro Production
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Bovine oocyte retrieval, IVM, IVF, and subsequent IVC up to the blastocyst stage were performed as previously described [23 (link)]. Briefly, cumulus-oocyte
complexes (COCs) collected from slaughterhouse-derived Holstein ovaries were cultured in TCM-199 medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 38.5°C in a humidified atmosphere
of 5% CO2 for 20–22 h. In vitro-matured oocytes were transferred to Brackett and Oliphant (B.O.) medium containing 2.5 mM theophylline (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque, Inc., Kyoto, Japan). Subsequently, freeze-thawed semen was centrifuged at 600 × g for 7
min in B.O. medium, and spermatozoa were added to the COCs at a final concentration of 5 × 106 cells/mL. The presumptive zygotes were denuded by pipetting after 12 h of incubation
and cultured in mSOFai medium at 38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 for eight days.
complexes (COCs) collected from slaughterhouse-derived Holstein ovaries were cultured in TCM-199 medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 38.5°C in a humidified atmosphere
of 5% CO2 for 20–22 h. In vitro-matured oocytes were transferred to Brackett and Oliphant (B.O.) medium containing 2.5 mM theophylline (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque, Inc., Kyoto, Japan). Subsequently, freeze-thawed semen was centrifuged at 600 × g for 7
min in B.O. medium, and spermatozoa were added to the COCs at a final concentration of 5 × 106 cells/mL. The presumptive zygotes were denuded by pipetting after 12 h of incubation
and cultured in mSOFai medium at 38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 for eight days.
5
Differentiation of Pancreatic Beta Cells
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Stage 7 medium is based on Rezania et al. with some modifications. Cells were exposed to MCDB 131 medium with 1% P/S, 2% fat-free BSA (FUJIFILM Wako), 14.44 mM glucose (to generate a final concentration of 20 mM), 1.5 g/L NaHCO3, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5 inhibitor II, 1 μM T3, 10 μM ZnSO4 (Merck Millipore), 1.4 IU/ml heparin sodium salt (Nacalai Tesque), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck), 1 μM PD-166866, 3 μM TR06141363, and 10 μM Y-27632, for 4 days.
6
Bovine oocyte-to-embryo production protocol
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Bovine oocyte retrieval, in vitro oocyte maturation (IVM), in vitrofertilization (IVF), and subsequent in vitro embryo culture (IVC) were performed as described previously [14] (link). Briefly, cumulus-oocyte complexes (COCs) collected from slaughterhouse-derived ovaries were matured by culturing in TCM-199 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 38.5 °C in a humidified atmosphere of 5% CO2 for 22-24 h. IVM oocytes were transferred to Brackett and Oliphant (B.O.) medium [15] (link) containing 2.5 mM theophylline (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 7.5 μg/ mL heparin sodium salt (Nacalai Tesque, Inc., Kyoto, Japan). Subsequently, frozenthawed semen was centrifuged at 600 × g for 7 min in the B.O. medium, and the spermatozoa were added to the COCs at a final concentration of 5 × 10 6 cells/mL. After 18 h of incubation, the presumptive IVF zygotes were denuded by pipetting and cultured in mSOFai medium [14] (link) at 38.5 °C in a humidified atmosphere of 5% CO2 and 5% O2. The start of insemination was regarded as day 0 (D0) after IVC.
7
Rapamycin-Induced Protein-Protein Interaction
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HEK293T cells were transfected with rapamycin-dimerization inducible pair (V5_LAR_FRBLgBiT/Myc_LAR_FKBPSmBiT) or rapamycin-dimerization uninducible pair (V5_LAR_LgBiT/Myc_LAR_SmBiT) so that the total DNA amount was 100 ng/well. After transfection, the cells were cultured at 37°C under 5% CO2 for about 22∼26 h. Then, before application of the substrates, the culture media were exchanged with Opti-MEM® I (1X) and incubated at ambient temperature for 10 min. The transfected wells were recorded for at least 5 min before substrate addition to assess the basal status. Luciferase substrate was applied for 5 min before the generated luminescence was recorded for 30 counts. Then, vehicle or rapamycin (20 nM) together with heparin [0 mg ml−1 (vehicle), 10 mg ml−1, 20 mg ml−1, 40 mg ml−1] (Heparin sodium salt Nacalai Tesque, catalogue number 17513-54) or CS [0 mg ml−1 (vehicle), 10 mg ml−1, 20 mg ml−1, 40 mg ml−1] (Sigma-Aldrich) were applied, and the subsequent changes in luminescence were recorded for 121 counts. The interval between counts was 1 min. The whole process was performed at ambient temperature. Luminescence was detected using a Centro XS3 LB960 High Sensitivity Microplate Luminometer.
8
Bovine IVF Embryo Development
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Frozen semen was thawed in warm water (38.5˚C) for 30 sec. Spermatozoa were washed by centrifugation at 600 g for 7 min in Brackett and Oliphant (BO) medium containing 2.5
mM theophylline (Wako Pure Chemical Industries, Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque). After removal of supernatant, the sperm pellet was diluted with IVF100
solution (Research Institute for the Functional Peptides, Yamagata, Japan) to make a final concentration of 5 × 106/ml of spermatozoa pellet. The matured COCs were transferred to
the prepared IVF100 sperm medium and cultured for 18 h at 38.5˚C in a humidified atmosphere of 5% CO2 and 5% O2 in air.
After fertilization, presumptive zygotes were denuded by pipetting to remove cumulus cells and then cultured in Bovine KSOMaa medium (Zenith Biotech, Guilford, UK) containing 5% FBS, 100
U/ml penicillin and 100 U/ml streptomycin at 38.5˚C in a humidified atmosphere of 5% CO2 and 5% O2 for 7 days. One-cell embryos were collected before starting IVC,
while 8-cell stage embryos, morulae, and expanded blastocysts were sampled on days 2, 5, and 7, respectively. In addition, expanded blastocysts on day 7 were carefully separated using a
micro-blade (Feather, Osaka, Japan) equipped with micromanipulator (Narishige, NY, USA) to obtain separated inner cell mass (ICM) with trophectoderm (TE) (ICM + TE) and TE alone (TE).
mM theophylline (Wako Pure Chemical Industries, Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque). After removal of supernatant, the sperm pellet was diluted with IVF100
solution (Research Institute for the Functional Peptides, Yamagata, Japan) to make a final concentration of 5 × 106/ml of spermatozoa pellet. The matured COCs were transferred to
the prepared IVF100 sperm medium and cultured for 18 h at 38.5˚C in a humidified atmosphere of 5% CO2 and 5% O2 in air.
After fertilization, presumptive zygotes were denuded by pipetting to remove cumulus cells and then cultured in Bovine KSOMaa medium (Zenith Biotech, Guilford, UK) containing 5% FBS, 100
U/ml penicillin and 100 U/ml streptomycin at 38.5˚C in a humidified atmosphere of 5% CO2 and 5% O2 for 7 days. One-cell embryos were collected before starting IVC,
while 8-cell stage embryos, morulae, and expanded blastocysts were sampled on days 2, 5, and 7, respectively. In addition, expanded blastocysts on day 7 were carefully separated using a
micro-blade (Feather, Osaka, Japan) equipped with micromanipulator (Narishige, NY, USA) to obtain separated inner cell mass (ICM) with trophectoderm (TE) (ICM + TE) and TE alone (TE).
9
Bovine In Vitro Fertilization Protocol
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Frozen semen was thawed in warm water (38.5°C) for 30 sec. Spermatozoa were washed by centrifugation at 600 g for 7 min in Brackett and Oliphant medium containing 2.5 mM
theophylline (Wako Pure Chemical Industries, Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque). After removing the supernatant, the sperm pellet was diluted with IVF100
solution (Research Institute for the Functional Peptides, Yamagata, Japan) to make a final concentration of 5 × 106/ml of spermatozoa pellet. The matured COCs were transferred to
IVF100 sperm medium and cultured for 18 h at 38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 in air. After fertilization, presumptive zygotes were denuded by
pipetting to remove cumulus cells and cultured in Bovine IVD101 medium (Research Institute for the Functional Peptides) containing 5% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin at
38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 in air for 8 days. The cleavage and blastocyst rates were recorded on day 2 and 8, respectively.
theophylline (Wako Pure Chemical Industries, Osaka, Japan) and 7.5 μg/ml heparin sodium salt (Nacalai Tesque). After removing the supernatant, the sperm pellet was diluted with IVF100
solution (Research Institute for the Functional Peptides, Yamagata, Japan) to make a final concentration of 5 × 106/ml of spermatozoa pellet. The matured COCs were transferred to
IVF100 sperm medium and cultured for 18 h at 38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 in air. After fertilization, presumptive zygotes were denuded by
pipetting to remove cumulus cells and cultured in Bovine IVD101 medium (Research Institute for the Functional Peptides) containing 5% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin at
38.5°C in a humidified atmosphere of 5% CO2 and 5% O2 in air for 8 days. The cleavage and blastocyst rates were recorded on day 2 and 8, respectively.
10
Bovine Oocyte Maturation and Fertilization
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Bovine oocyte retrieval, in vitro oocyte maturation, fertilization and subsequent in vitro embryo culture were performed as described previously (Nagatomo et al. 2013) (link). Briefly, cumulus-oocyte complexes (COCs) collected from slaughterhouse-derived ovaries were matured by culturing in TCM-199 medium (Thermo Fisher Scientific) at 38.5°C in a humidified atmosphere of 5% CO 2 in air for 20-22 h. In vitromatured oocytes were transferred to Brackett and Oliphant (BO) medium (Brackett & Oliphant 1975) (link) containing 2.5 mM theophylline (Wako Pure Chemical Industries) and 7.5 μg/ mL heparin sodium salt (Nacalai Tesque, Inc., Kyoto, Japan). Subsequently, frozen-thawed semen was centrifuged at 600 g for 7 min in BO medium, and the spermatozoa were added to the COCs at a final concentration of 5 × 10 6 cells/mL. After 12 h of incubation, the presumptive zygotes were denuded by pipetting and cultured in mSOFai medium (Aono et al. 2013) (link) at 38.5°C in a humidified atmosphere of 5% CO 2 and 5% O 2 in air for 8 days.
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