Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods

Artificial insemination with frozen-thawed spermatozoa is commonly used in cattle breeding. A simple and fast procedure is needed for routine evaluation of the acrosomal status of frozen-thawed bovine sperm. Therefore, the purpose of this study was to test two staining procedures used to determine the viability and integrity of acrosome of frozen-thawed bovine spermatozoa. Double staining and Hoechst/FITC- Pisum sativum agglutinin (FITC-PSA) labelling were tested for evaluating the viability and acrosome reaction induced by calcium ionophore of bull spermatozoa. In our experiments no significant differences were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%). The viability of sperm stained by the double staining method was 67.17%, and a higher portion of viable sperm (82.67%) was observed by staining with the Hoechst procedure (P < 0.01). On the basis of the results obtained it is concluded that both methods can be used for detecting the acrosome reaction of frozen-thawed bovine spermatozoa

Tetraspanins, More than Markers of Extracellular Vesicles in Reproduction

The participation of extracellular vesicles in many cellular processes, including reproduction, is unquestionable. Although currently, the tetraspanin proteins found in extracellular vesicles are mostly applied as markers, increasing evidence points to their role in extracellular vesicle biogenesis, cargo selection, cell targeting, and cell uptake under both physiological and pathological conditions. In this review, we bring other insight into the involvement of tetraspanin proteins in extracellular vesicle physiology in mammalian reproduction. We provide knowledge regarding the involvement of extracellular vesicle tetraspanins in these processes in somatic cells. Furthermore, we discuss the future direction towards an understanding of their functions in the tissues and fluids of the mammalian reproductive system in gamete maturation, fertilization, and embryo development; their involvement in mutual cell contact and communication in their complexity

Quality evaluation of commercial anti-Salmonella antibodies for immunomagnetic separation using whole-cell dot blot

The selection of antibodies with the desired specificity, immunoreactivity, purity and integrity is a key step in developing an effective immunosorbent for immunomagnetic separation (IMS). The choice of methods for characterizing the antibodies is limited, especially with antibodies specific to surface cell structures. In this study, four commercial anti-Salmonella antibodies were evaluated from the point of view of their immunoreactivity with the cells of Salmonella Typhimurium and of their purity. For these purposes, traditional SDS-PAGE analysis with subsequent silver staining and a newly adapted whole-cell dot blot technique were applied. Based on this testing, monoclonal anti-core LPS Salmonella antibodies from MyBiosource unambiguously demonstrated the highest immunoreactivity to Salmonella Typhimurium cells, whereas cross-reactivity with the closely related G- bacteria, Escherichia coli and Citrobacter braakii was not observed. Such antibodies will be subsequently used for the preparation of an anti-Salmonella immunosorbent and applied in IMS. The immunoreactivity and selectivity of anti- Salmonella poly- and monoclonal antibodies are also discussed

Kvalitativní hodnocení komerčně dostupných anti-salmonelových protilátek pro imunomagnetickou separaci pomocí dot-blot analýzy celých buněk

The selection of antibodies with the desired specificity, immunoreactivity, purity and integrity is a key step in developing an effective immunosorbent for immunomagnetic separation (IMS). The choice of methods for characterizing the antibodies is limited, especially with antibodies specific to surface cell structures. In this study, four commercial anti-Salmonella antibodies were evaluated from the point of view of their immunoreactivity with the cells of Salmonella Typhimurium and of their purity. For these purposes, traditional SDS-PAGE analysis with subsequent silver staining and a newly adapted whole-cell dot blot technique were applied. Based on this testing, monoclonal anti-core LPS Salmonella antibodies from MyBiosource unambiguously demonstrated the highest immunoreactivity to Salmonella Typhimurium cells, whereas cross-reactivity with the closely related G- bacteria, Escherichia coli and Citrobacter braakii was not observed. Such antibodies will be subsequently used for the preparation of an anti-Salmonella immunosorbent and applied in IMS. The immunoreactivity and selectivity of anti-Salmonella poly- and monoclonal antibodies are also discussed.Klíčovým krokem při přípravě imunosorbentu pro efektivní imunomagnetickou separaci buněk je výběr protilátek s požadovanou specifitou, imunoreaktivitou a čistotou. Jejich charakterizace z pohledu těchto vlastností je obtížná, zejména pokud se jedná o protilátky proti povrchových buněčným strukturám. Tato studie se zabývá kvalitativním hodnocení čtyř komerčně dostupných anti-salmonelových protilátek z pohledu jejich čistoty a imunoreaktivity na buňky bakterie Salmonella Typhimurium. Ke kvalitativní analýze vybraných protilátek byla použita tradiční metoda SDS-PAGE a nově adaptovaná dot-blot technika pro celé buňky. Výsledky této studie ukazují nejvyšší imunoreaktivitu monoklonálních anti-Salmonella protilátek od firmy MyBiosource. Zkřížená reaktivita s G- bakteriemi Escherichia coli a Citrobacter braakii nebyla pozorována