The Annexin V-FITC Apoptosis Detection kit was purchased from KeyGEN bioTECH Company (Nan Jing, Jiang Su, China). Human Tubal Fluid (HTF) was obtained from Millipore Co., Ltd. (Bedford, MA, USA). ROS, TAC (ABTS method), and bicinchoninic acid (BCA) kits were provided by Nanjing Jiancheng Bioengineering Institute (Nan Jing, Jiang Su, China). Polyclonal anti-cytochrome C, anti-caspase-3, anti-bcl-2, and anti-bax antibodies were purchased from Cell Signaling Technology Inc. (Danvers, Massachusetts, USA). Goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate and enhanced chemiluminescence (ECL) kit were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). PVDF membranes were purchased from Merck Millipore Inc. (Danvers, Massachusetts, USA). The mitochondrial protein extract kit was purchased from Beyotime Ltd. (Haimen, China). Trizol (Invitrogen, USA) was used for RNA extraction. The Ex TaqTM PCR kit was purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China). SYBR Green/Flourescein qPCR Master Mix(2X) was purchased from Thermo Scientific Fermantas (USA).
Human tubal fluid
Manufactured by Merck Group
Sourced in United States, France
Human Tubal Fluid is a laboratory product designed to simulate the natural fluid environment of the human fallopian tube. It is a sterile, cell-culture tested solution that replicates the physiochemical properties of human tubal fluid. The product is intended for use in assisted reproductive technology (ART) research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (2 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti bax antibody, by Cell Signaling Technology (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Mitochondrial protein extract kit, by Beyotime (1 mentions)
M2 medium, by Merck Group (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Eclipse ti u inverted microscope, by Nikon (1 mentions)
Sperm class analyzer, by Microptic (1 mentions)
7 protocols using human tubal fluid
1
Apoptosis Assay in Human Tubal Fluid
2
Cauda Epididymal Sperm Isolation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following dissection from the body cavity, the two cauda epididymis were dissected and nicked three times to allow sperm to swim out. Cauda were placed in 1ml of Human Tubal Fluid (HTF) (Millipore) at 37°C, and rotated in a 37°C incubator for 10 minutes. Cauda were removed and a 100μl aliquot was used for pre-swim-up baseline sperm counts. The remaining portion of sperm in HTF was then removed and placed in a new tube using wide bore tips. Sperm were centrifuged for 5 minutes at 400xg and the supernatant discarded. The pellet was re-suspended in 1ml of fresh 37°C HTF and centrifuged for 5 minutes at 400xg. The supernatant was removed and 1ml of fresh 37°C HTF was carefully overlaid on top of the pellet. The tube was then placed at a 45° angle in a 37°C incubator for 1 hour; after which the top 800μl containing motile sperm was removed and placed in a new tube. All aliquots of sperm used for counting were diluted 1:10 in H2O and counted using a hemocytometer. Counts were performed blind with four technical replicates per mouse. Sperm counts were calculated by taking the average number of sperm from each of the four technical replicates per mouse. Percent motility was calculated by dividing the post swim-up count by the pre-count and multiplying by 100. All analyses between groups were performed with an unpaired two-tailed student's t-test, unless otherwise noted.
3
Sperm Collection from Rat and Human
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat sperm cell collection. After sacrifice, we collected and cut the epididymis to enable the exit of sperm in HTF-BSA culture medium (Human Tubal Fluid, Millipore®, France, with 0.4% BSA: Bovine Serum Albumin, Sigma-Aldrich®, Lyon, France) for 1 h at 37 °C and CO2 5%.
Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 min at 420 g. The supernatants were discarded, and the pellets were exposed to various exposure conditions.
Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 min at 420 g. The supernatants were discarded, and the pellets were exposed to various exposure conditions.
4
Isolation and Preparation of Rat and Human Gametes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat cumulus–oocytes complex (COC) collection. Female superovulation was induced in prepubescent rats by an intraperitoneal injection of pregnant mare serum gonadotropin (20 U.I. PMSG) on day one and human chorionic gonadotropin (40 U.I. HCG) on day three. Twelve hours later, we collected oviducts containing oocytes surrounded by follicle cells after cervical dislocation euthanasia [51 (link)]. Once the cells from each oviduct were recovered, we left them equilibrate in Ferticult® medium at 37 °C and CO2 5% for 1 h [46 (link)].
Rat sperm cell collection. Male rats were previously anesthetized (Sevoflurane, vol % 8) and then euthanized with a 10 mL injection of Dolethal. After sacrifice, we collected and cut the epididymis to allow the exit of sperm into HTF-BSA culture medium (Human Tubal Fluid, Millipore, St-Quentin-en-Yvelines, France, with 0.4% BSA: Bovine Serum Albumin, Sigma-Aldrich, St. Quentin-Fallavier, France) for 1 h at 37 °C and CO2 5% under mineral oil (Sigma-Aldrich®, France) [30 (link)].
Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 minutes at 420× g. The supernatants were discarded, and the pellets were exposed to various exposure conditions [30 (link)].
Rat sperm cell collection. Male rats were previously anesthetized (Sevoflurane, vol % 8) and then euthanized with a 10 mL injection of Dolethal. After sacrifice, we collected and cut the epididymis to allow the exit of sperm into HTF-BSA culture medium (Human Tubal Fluid, Millipore, St-Quentin-en-Yvelines, France, with 0.4% BSA: Bovine Serum Albumin, Sigma-Aldrich, St. Quentin-Fallavier, France) for 1 h at 37 °C and CO2 5% under mineral oil (Sigma-Aldrich®, France) [30 (link)].
Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 minutes at 420× g. The supernatants were discarded, and the pellets were exposed to various exposure conditions [30 (link)].
5
IVF Protocol for Superovulated Mouse Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IVF experiments were performed essentially as described (Sharma et al., 2016 (link)) with some modifications. Briefly, female C57BL/6J mice (age 6–8 weeks) were superovulated by intraperitoneal injection of pregnant mare's serum gonadotropin (PMSG; 5 U; Calbiochem) followed by human chorionic gonadotropin (hCG; 5 U; Sigma-Aldrich) 48 hr later (Yamashita et al., 2008 (link)). Metaphase II-arrested oocytes tightly packed with cumulus cells were collected from the oviductal ampulla 14 hr after hCG injection and placed in a 100 μl drop of human tubal fluid (HTF; Millipore) medium covered with mineral oil. Fresh cauda epididymal sperm of fl/Y and cKO/Y littermates (>6 animals per genotype both for Sox2-Cre and Ngn3-Cre, aged 2–4 mo) were swum-up in 1 ml HTF medium for 10 min and capacitated by incubation for another 30 min at 37°C under 5% CO2. An aliquot (1.0 × 105 cells) of the capacitated sperm suspension was added to 100 μl drop of HTF medium containing the oocytes. After incubation at 37°C under 5% CO2 for 4 hr, the presumed zygotes were washed with KSOM medium to remove cumulus cells, sperm and debris, and then incubated in a 50 μl drop of KSOM medium. In vitro fertilized embryos were analyzed at cleavage (24 hr) and blastocyst stages (96 hr).
6
Histological Analysis of Mouse Reproductive Organs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological analyses of mouse testis and epididymis were carried out as described previously21 (link). Briefly, tissues were dissected and fixed in 10% formalin solution. Fixed tissues were then embedded in paraffin, sectioned and H & E stained by the Histopathology laboratory at Michigan State University. H & E staining was examined under a light microscope (Nikon Eclipse E600) by S.G, H.X., and G.I.P who is a certified veterinarian with extensive experience in mouse reproduction research. For the examination of epididymal sperm, cauda epididymides were collected in M2 medium (Sigma-Aldrich, MO, USA) and immediately transferred into HTF medium (Human Tubal Fluid, EMD Millipore, MA, USA) followed by incubating at 37 °C, 5% CO2 for 1 hour. An aliquot of sperm suspension (10 µl) was fixed and examined under a phase contrast microscope (Nikon Eclipse Ti-U inverted microscope).
7
Evaluating Circadian Desynchrony Effects on Sperm
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After sacrifice, both epididymides of all circadian desynchrony and control mice were isolated, cleared of adhering tissues, chopped into pieces, and incubated in 800 μl of human tubal fluid (Merck, USA) at 37°C to allow sperm to spread into the medium. An aliquot of the mixture (10 μl) was analyzed for sperm concentration and progressive motility using a Sperm Class Analyzer (Microptic S.L., Barcelona, Spain). The sperm concentration per μl was multiplied by 800 to obtain total sperm count. Researchers performing these analyses were blinded to animal group allocation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!