Humid incubator

Manufactured by Astec

Sourced in Japan

The Humid Incubator is a laboratory equipment designed to provide a controlled environment for the cultivation and growth of various biological specimens. It maintains a consistent temperature and humidity level within the incubation chamber, which is essential for the optimal development of cell cultures, microorganisms, and other samples.

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5% CO2 humid incubator (2 citations)

5 protocols using humid incubator

Porcine Oocyte Maturation with GT1b

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Porcine ovaries were collected from a slaughterhouse. Porcine follicular fluid (pFF) and cumulus-oocyte
complexes (COCs) were recovered from 3–6 mm ovarian follicles using an aspiration method. The medium used
during IVM was composed of TCM199 (Gibco, Life Technologies, Melbourne, Australia), 0.6 mM cysteine, 0.91 mM
sodium pyruvate, 10 ng/m epidermal growth factor, 75 μg/ml kanamycin, 1 μg/ml insulin, 10% (v/v) and pFF. GT1b
was used to treat the oocytes during IVM at concentrations of 0 μM, 5 μM, 10 μM and 20 μM. Porcine COCs were
co-cultured at 50 to 60 cells per well using a 4-well dish (Nunc, Roskilde, Denmark) with 500 μl of IVM medium
(0 μM group). 20 μl of DI water was added to all groups. IVM was performed at 39 C in 5% CO₂ using
a humid incubator (Astec, Fukuoka, Japan). Maturation was performed in IVM media with 10 IU/ml equine
chorionic gonadotropin (eCG) and 10 IU/ml human chorionic gonadotropin (hCG) for 22 h. The cells were then
moved into hormone-free IVM medium and cultured for 18 h. Matured COCs were denuded by gentle pipetting with
0.1% hyaluronidase and TLH-PVS medium. Cumulus cells and denuded oocytes were obtained through this process
and then used for subsequent experiments.

HWANG S.U., JEON Y., YOON J.D., CAI L., KIM E., YOO H., KIM K.J., PARK K.M., JIN M., KIM H, & HYUN S.H. (2015). Effect of ganglioside GT1b on the in vitro maturation of porcine oocytes and embryonic development. The Journal of Reproduction and Development, 61(6), 549-557.

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Isolation and Culture of Fetal Fibroblasts

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Ear tissues separated from stillborn pigs (Sus scrofa domesticus) and dogs (Canis lupus familiaris) were washed in Dulbecco’s phosphate-buffered saline-positive containing 1% (v/v) antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Ear tissues were sterilized in 70% ethanol and washed with Dulbecco’s phosphate-buffered saline-positive. Skin tissues were separated from sterilized ear tissue and chopped finely using a blade. Chopped skin tissues were incubated with 0.25% trypsin for 2 h and then added to the cell culture medium [Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 2% (v/v) antibiotic-antimycotic (Gibco)] to neutralize the trypsin. Trypsinized tissues were cultured in a 100-mm culture dish and adherent cells were subcultured. The incubation conditions for primary culture were 37 °C and 5% CO₂ in a humid incubator (Astec, Fukuoka, Japan). Dog and pig fetal fibroblast cells were maintained in DMEM supplemented with 10% FBS, 1% Glutamax (Gibco), 1% non-essential amino acids (Gibco), 1% antibiotic-antimycotic, and 0.1% 2-mercaptoethanol (Gibco).

Eun K., Hwang S.U., Jeong Y.W., Seo S., Lee S.Y., Hwang W.S., Hyun S.H, & Kim H. (2017). SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo. Biological Procedures Online, 19, 13.

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Porcine Oocyte Collection and In Vitro Maturation

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The ovaries of prepubertal gilts obtained from a local abattoir were transported to the laboratory at 34–37°C. Cumulus oocyte complexes (COCs) were aspirated from follicles of 3–8 mm in diameter. COCs with multiple layers of compacted cumulus cells were selected and washed three times in HEPES-buffered Tyrode’s medium (TLH) containing 0.05% (w/v) PVA (TLH‐PVA). COCs were placed in each well of a four-well multi-dish (Nunc, Roskilde, Denmark). Each well contained 500 µL of IVM medium with 10 IU/ml equine chronic gonadotropin and 10 IU/ml human chorionic gonadotropin (Intervet, Boxmeer, Netherland). After 22 h, the COCs were transferred to a fresh IVM medium without eCG/hCG and incubated for 22 h. IVM was performed at 39°C and 5% CO₂ in a humid incubator (Astec, Fukuoka, Japan). After IVM, COCs were denuded by gentle pipetting with 0.1% hyaluronidase.

Lee J., Cai L., Kim M., Choi H., Oh D., Jawad A., Lee E, & Hyun S.H. (2022). Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines. Frontiers in Cell and Developmental Biology, 10, 948778.

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In Vitro Maturation of Porcine Oocytes

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Porcine ovaries obtained from a slaughterhouse were delivered to the laboratory in a thermos containing 0.9% saline and at 36 °C. Within 2 h, 3–6-mm ovarian follicles were aspirated using a 10-mL syringe with an 18-gauge needle. The obtained porcine follicular fluid was centrifuged at 500×g for 30 min at 4 °C, and then filtered through 0.45-μm filters. Cumulus-oocyte complexes (COCs) were washed using TLH-PVA medium [HEPES-buffered Tyrode’s medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (PVA)], and then co-cultured with 50–60 COCs per well in a 4-well dish (Nunc, Roskilde, Denmark) containing 500 μL of IVM medium. IVM was performed in IVM medium containing 10 IU/mL equine chorionic gonadotropin and 10 IU/mL human chorionic gonadotropin for 22 h, and then the cells were moved to hormone-free IVM medium and cultured for 18 h. The composition of IVM medium was TCM199 (Gibco), 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 75 μg/mL kanamycin, 1 μg/mL insulin, and 10% (v/v) porcine follicular fluid. The incubation conditions for IVM were 39 °C and 5% CO₂ in a humid incubator (Astec). Mature COCs were denuded by gently pipetting with 0.1% hyaluronidase, and then washed with TLH-PVA medium. MII oocytes obtained by this process were used for subsequent experiments.

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Porcine Oocyte Maturation Protocol

Cited 1 time

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Oocyte collection and in vitro maturation was performed according to Hwang et al. [18 (link)]. Briefly, porcine ovaries were collected from a slaughterhouse. Porcine follicular fluid (pFF) and cumulus-oocyte complexes (COCs) were recovered from 3 to 6 mm ovarian follicles by aspiration. The composition of the medium used during in vitro maturation (IVM) was as follows: TCM199 (Gibco), 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL EGF, 75 µg/mL kanamycin, 1 µg/mL insulin, and 10% (v/v) pFF. Porcine COCs were co-cultured at 50 to 60 cells per well in a 4-well dish (Nunc, Denmark) with 500 µL IVM media. The conditions for IVM were 39℃ in a 5% CO₂ atmosphere in a humid incubator (Astec, Japan). Maturation was performed in IVM medium with 10 IU/mL equine chorionic gonadotropin and 10 IU/mL human CG for 22 h. The cells were then moved into hormone-free IVM medium and cultured for 18 h. Matured COCs were denuded by using gentle pipetting with 0.1% hyaluronidase and HEPES-buffered Tyrode's medium containing 0.05% (w/v) polyvinyl alcohol (TLH-PVA) medium. Denuded oocytes obtained through this process were used in subsequent experiments.

Hwang S.U., Eun K., Yoon J.D., Kim H, & Hyun S.H. (2018). Production of transgenic pigs using a pGFAP-CreERT2/EGFPLoxP inducible system for central nervous system disease models. Journal of Veterinary Science, 19(3), 434-445.

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