Human tubal fluid

Manufactured by Irvine Scientific

Sourced in United States

Human tubal fluid (HTF) is a culture medium designed to mimic the natural environment of the human fallopian tube. It provides a balanced salt solution, energy sources, and pH buffering to support the growth and development of cells, such as embryos, in an in vitro setting.

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Lab products found in correlation

Human chorionic gonadotropin (hcg), by Merck Group (2 mentions) Hyaluronidase, by Merck Group (2 mentions) Human serum albumin (hsa), by Irvine Scientific (1 mentions) In situ cell death detection kit with fluorescein, by Roche (1 mentions) Triton x 100, by Merck Group (1 mentions) Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions) Stereomicroscope, by Zeiss (1 mentions) Bsa fraction 5 fatty acid free, by Merck Group (1 mentions) Epidermal growth factor (egf), by PanEco (1 mentions) Follicle stimulating hormone, by Merck Group (1 mentions) Doxorubicin dox, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

7 protocols using human tubal fluid

Fused Silica Capillary Vitrification Protocol

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Human tubal fluid (HTF) and human serum albumin (HSA) solutions were purchased from Irvine Scientific (Santa Ana, CA, USA). Trehalose was obtained from Ferro Pfanstiehl (Waukegan, IL, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
Fused silica capillary tubes coated with a thin layer of polyimide were obtained from Postnova Analytics Inc. (UT, USA). Each micro-capillary was 6.5 cm in length, 200 μm in inner diameter, and 2 μl in capacity. Polyimide has a thermal conductivity about 6.5 times less than the fused silica (fused silica ~1.35 Wm⁻¹K⁻¹ [6 ]; polyimide ~0.2 Wm⁻¹K⁻¹ [20 (link),23 ]). Heo et al. [13 (link)] have successfully used fused silica capillary tubes, after removing the polyimide coat, in which to vitrify a variety of cell types. Part of their success is attributed to the high thermal conductivity of the wall of the fused silica tubes which allows very rapid cooling [22 (link)]. The polyimide was not removed in our work.

Liu J., Tanrikut C., Wright D.L., Lee G.Y., Toner M., Biggers J.D, & Toth T.L. (2016). Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant. Cryobiology, 73(2), 162-167.

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Comprehensive IVF/ICSI Procedure Protocol

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The details for ovarian stimulation, oocyte retrieval, IVF/ICSI procedures, and embryo culture have been described elsewhere (Huang et al., 2019 (link)). hMG (Anhui Fengyuan Pharmaceutical) was started on day 3 of the menstrual cycle. Urinary hCG (Lizhu Pharmaceutical Trading Co., China) was administered at a dose of 5,000 IU when two or more follicles measured 18 mm or more. Serum FSH, LH, E₂, and P levels were analyzed on spontaneous menstrual cycle day 3 (MC₃) and the day of hCG triggering. Oocytes retrieval was scheduled at 34–36 h after trigger and IVF or ICSI was performed 4–5 h after oocytes retrieval.
For IVF, collected oocytes were incubated in human tubal fluid (HTF; Irvine Scientific, United States), supplemented with 10% serum substitute supplement (SSS; Irvine Scientific, United States), and 300,000 progressively motile spermatozoa and left overnight; For ICSI, denudated oocytes were injected with a single mechanically immobilized sperm and directly thereafter cultured in fertilization medium (HTF + 10% SSS). All embryos were maintained in Continuous Single Culture of HTF (Irvine Scientific, United States) and were incubated under oil at 37°C and a 5% O₂ and 6% CO₂ humidified incubators with 30 μL of culture media drop throughout the entire duration of in vitro culture.

Zhao L., Guan Y., Meng Q., Wang W., Wu L., Chen B., Hu J., Zhu J., Zhang Z., Mu J., Chen Y., Sun Y., Wu T., Wang W., Zhou Z., Dong J., Zeng Y., Liu R., Li Q., Du J., Kuang Y., Sang Q, & Wang L. (2021). Identification of Novel Mutations in CDC20: Expanding the Mutational Spectrum for Female Infertility. Frontiers in Cell and Developmental Biology, 9, 647130.

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Assessment of Sperm DNA Fragmentation

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Post-thawed spermatozoa were washed in Human Tubal Fluid (HTF) (Irvine Scientific) supplemented with 10% Synthetic Serum Substitute (SSS) (Irvine Scientific) and then with Phosphate Buffered Saline (PBS) 1× (Sigma-Aldrich) and centrifuged at 500 ×g for 5 min. After that, sperm samples were washed and resuspended in PBS solution at a final concentration of 20×10⁶/mL; and fixed in methanol 80%. Then slides were washed and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) (In situ cell death detection kit with fluorescein, Roche Diagnostics, Mannheim, Germany) was performed to detect sperm DFI, according to the manufacturer’s protocol. To inhibit photobleaching of fluorescent dyes, there was the addition of VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). At least 200 cells were analyzed per slide using epifluorescent microscopy (34 (link)-36 (link)). Negative and positive controls (no TdT enzyme and treatment with DNAase, respectively) were performed in each experiment. The TUNEL negative spermatozoa fluoresced blue (spermatozoa without fragmented DNA), whereas the TUNEL-positive spermatozoa fluoresced bright green (spermatozoa with fragmented DNA). The final percentage of spermatozoa with fragmented DNA was reported for each sample.

Sicchieri F., Silva A.B., Santana V.P., Vasconcelos M.A., Ferriani R.A., Vireque A.A, & dos Reis R.M. (2021). Phosphatidylcholine and L-acetyl-carnitine-based freezing medium can replace egg yolk and preserves human sperm function. Translational Andrology and Urology, 10(1), 397-407.

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Oocyte Collection and Maturation Analysis

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Ovulation was induced by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG, 10 IU; Sigma-Aldrich) followed by human chorionic gonadotropin (hCG, 10 IU; Sigma-Aldrich) 46–48 h later. Fifteen to sixteen hours post hCG injection, mice were killed by CO₂ asphyxiation, and the ovaries and attached oviducts were collected. Oocytes were released from the oviducts by puncturing the oviducts with an insulin syringe, and oocytes were collected via pulled glass pipet. Collected oocytes were denuded of cumulus cells by incubating for 2 min in 80 IU/mL of hyaluronidase (Sigma-Aldrich) at 37°C, followed by three washes with human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with 0.4% BSA (fraction V, fatty acid free; Sigma-Aldrich) at 37°C. Oocytes were counted and classified using a Zeiss Stereo Microscope (Zeiss) as MII (extrusion of the first polar body into the perivitelline space), maturation arrested (germinal vesicle stage, or germinal vesicle breakdown without polar body extrusion) or dead (membrane blebbing, oocyte fragmentation or condensed cytoplasm). Following analysis, MII oocytes were fixed in 2% paraformaldehyde for 30 min at 37°C for endpoint analyses.

Faraci C., Annis S., Jin J., Li H., Khrapko K, & Woods D.C. (2018). Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse. Reproduction (Cambridge, England), 156(2), 185-194.

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Comprehensive Cell Culture Protocol

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All the laboratory culture dishes used for the current study were purchased from SPL, Lifesciences unless stated otherwise. The dishes used for molecular analysis were purchased from Axygen (Corning, USA). Insulin-transferrin-selenium, phosphate buffer saline (PBS), epidermal growth factor (EGF) and Minimum Essential Medium Eagle–Alpha Modification (α-MEM) were purchased from PanEco (Russia). Fetal calf serum (FCS) was purchased from Hyclone (Thermo-Fischer, USA). Human Tubal Fluid was purchased from Irvine Scientific (USA). Gentamycin and ascorbic acid were purchased from Dalhimfarm (Russia). Human chorionic gonado-tropin (hCG) and follicle stimulating hormone (FSH) were purchased from Merck Serono (Switzerland). All other reagents were purchased from Sigma-Aldrich (USA) unless stated otherwise.

Filatov M.A., Nikishin D.A., Khramova Y.V, & Semenova M.L. (2020). The in vitro Analysis of Quality of Ovarian Follicle Culture Systems Using Time-Lapse Microscopy and Quantitative Real-Time PCR. Journal of Reproduction & Infertility, 21(2), 94-106.

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Rat Epididymal Sperm Analysis

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The left epididymis tails of F1 and F2 male rats were placed in 2 ml of 37°C normal saline (pre-warm required), chopped, and placed for two minutes. Next, 100 μL of sperm suspension was added into 1.0 ml of modified HTF medium (Human Tubal Fluid, Irvine Scientific) and incubated at 37°C for two minutes, 10 μl of which was applied to the preheated blood cell counting plate for sperm quality analysis (25 (link)).
Sperm density, mobility and morphology were evaluated. For sperm density, six fields of view were selected and the counting was completed in 2 min. Sperm motility evaluation was performed by the same person throughout the study and was assessed by the computer-aided sperm analysis (CASA) with a phase-contrast microscope (Leica DMLS) at ×200 magnification. Sperm morphology was also evaluated, the spermatozoa were categorized in wet preparations using phase contrast optic. A spermatozoon with a rudimentary tail or a round or detached head was considered morphologically abnormal.

Zhang H.L., Yi M., Li D., Li R., Zhao Y, & Qiao J. (2020). Transgenerational Inheritance of Reproductive and Metabolic Phenotypes in PCOS Rats. Frontiers in Endocrinology, 11, 144.

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Doxorubicin-Induced Oocyte Fragmentation

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Female mice (3-4 months old) were superovulated with 10 IU pregnant mare serum gonadotropin (PMSG) (Calbiochem, CA), followed 46h later by injection with 7.5 IU Human Chorionic Gonadotropin (hCG) (Calbiochem, CA). Mature oocytes were collected 16h following hCG injection. Cumulus cells were removed by brief incubation in 80 IU hyaluronidase (Sigma, MO). Oocytes were cultured throughout the experiment in 0.2 mL of human tubal fluid (Irvine Scientific, CA) supplemented with 0.5% bovine serum albumin (Sigma, MO) under mineral oil at 37°C and 5% CO₂. Oocytes were treated with 200 nM or 1 μ M Doxorubicin (DOX) (Sigma, CA) or DMSO (Control) for 24h and were analyzed for cytoplasmic fragmentation every 2h. Following treatment, oocytes were stained with DAPI and further inspected. The eEF2K +/+ oocyte sample size was 16-24 oocytes per treatment group, and the eEF2K −/− oocyte population size was 18-19 oocytes per treatment group.

Chu H.P., Liao Y., Novak J.S., Hu Z., Merkin J.J., Shymkiv Y., Braeckman B.P., Dorovkov M.V., Nguyen A., Clifford P.M., Nagele R.G., Harrison D.E., Ellis R.E, & Ryazanov A.G. (2014). Germline quality control: eEF2K stands guard to eliminate defective oocytes. Developmental cell, 28(5), 561-572.

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