Sperm cells were extracted from a modified two-gradient (50%–80%) Percoll centrifugation method from ejaculated human semen obtained from nine genotyped men.11 (link) The sperm pellet was washed three times and resuspended in phosphate buffered saline, and then reanalyzed with a Sperm Class analyzer system (Microptic S.L., Barcelona, Spain). The sperm pellet (99% purity) was resuspended in denaturing lysis buffer (20 mmol l–1 Tris, pH 7.4, 2% sodium dodecyl sulfate and 1 mM phenylmethanesulfonyl fluoride). The supernatant was collected after centrifugation and protein was quantified by the bicinchoninic acid assay. Total protein quantity was dependent on sperm number because of purity (99%). Twenty micrograms of protein obtained from each patient was loaded into each lane for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane using an electrophoretic method (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The human MCT2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Sperm class analyzer system
Manufactured by Microptic
Sourced in Spain
The Sperm Class analyzer system is a lab equipment product designed to assess and analyze sperm samples. It provides objective measurements and data on various sperm parameters such as concentration, motility, and morphology.
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Lab products found in correlation
4 protocols using sperm class analyzer system
1
Sperm Protein Extraction and Analysis
2
Semen Analysis and Sperm Purification
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Semen variables were examined after liquefaction according to the World Health Organization laboratory manual [19 (link)]. Semen volume was measured with a 10 mL serological pipet. The Sperm Class Analyzer system (Microptic S.L., Barcelona, Spain) was employed to determine sperm concentration (×106/mL), sperm motility (%), and straight-line velocity (VSL) (μm/s). Semen smears stained with Berg's stain (carbol fuchsin and methylene blue) were used to detect sperm abnormalities. After semen analysis, the remaining raw semen was filtered through glass wool to remove gelatinous material, and then, sperm was obtained by centrifugation twice at 1000 g for 10 minutes (min) and resuspension in Earle's balanced salt solution (EBSS). Finally, spermatozoa were purified by the swim-up method (SUM).
3
Sperm Hyperactivation Analysis Protocol
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After 90 min-capacitation in the presence of CCCP (concentration range: 10–60 μM), H89 (20 μM) or DMSO, sperm aliquots (15 μl) were placed between pre-warmed slides and cover slips (22 × 22 mm) to create a chamber with 30 μm depth, and were examined at 37°C using Sperm Class Analyzer® system (SCA v.6.2.0.1., Microptic SL, Barcelona, Spain). Drifting was set in 25 μm/s. At least 200 sperm distributed in a minimum of 10 different microscope fields were evaluated (30 frames acquired at 60 Hz for each measurement). The following parameters were assessed: curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN,%), straightness (STR,%), wobble (WOB,%), amplitude of lateral head displacement (ALH, μm) and beat cross frequency (BCF, Hz). Sperm were considered hyperactivated when presenting VCL ≥ 271 μm/s, LIN < 50% and ALH ≥ 3.5 μm. These custom cutoffs were selected based on our experience (Brukman et al., 2016 (link)) and previously reported recommendations (Bray et al., 2005 (link)).
4
Semen Analysis Using Sperm Class Analyzer
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Semen samples were transferred in a water bath at 18 C to the laboratory of Germplasm Conservation and Reproductive Biotechnologies at the Agricultural Research Council (ARC) in Irene. The time taken from the collection site to the laboratory at ARC was 30 min. The sperm total motility (%), speed and progression of the sperm were determined using the Sperm Class Analyzer® system (Microptic, Spain). Semen samples were diluted with Tris-hydroxymethyl aminomethane (1:100) and 5 L were placed on a pre-warmed glass slide and then mounted with a glass cover-slip before evaluation at a magnification of 10× (Nikon®, Shanghai, China).
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