Sperm viability was assessed by checking the membrane integrity using two separate fluorochromes SYBR-14 and PI (LIVE/DEAD Sperm Viability Kit; Molecular Probes, Invitrogen, Milan, Italy). SYBR-14 is a membrane-permeable dye, which stains the head of viable spermatozoa in green, while PI is a membrane-impermeable dye that only penetrates through disrupted plasma membrane, staining the sperm heads of non-viable cells in red. Sperm samples were diluted with BTS to a concentration of 1 × 106 spermatozoa/mL and aliquots of 500 μL were stained with 5 μL SYBR-14 working solution (final concentration: 100 nM) and with 2.5 μL of PI (final concentration: 12 μM) for 10 min at 37°C in darkness. Viable spermatozoa exhibited a positive staining for SYBR-14 and negative staining for PI (SYBR-14+/PI-). Single-stained samples were used for setting the voltage gain for FL1 and FL3 photomultipliers.
Live dead sperm viability kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy, China, Germany
The LIVE/DEAD Sperm Viability Kit is a fluorescence-based assay used to assess the viability of sperm cells. It utilizes two nucleic acid stains to differentiate between live (intact cell membrane) and dead (compromised cell membrane) sperm cells.
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Lab products found in correlation
Cytoflex cytometer, by Beckman Coulter (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Facscalibur, by BD (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (2 mentions)
Methanol, by Merck Group (2 mentions)
Ds fi2, by Nikon (2 mentions)
Ch 30 rf 200, by Olympus (2 mentions)
Eclipse epifluorescence microscope, by Nikon (2 mentions)
Fitc pna, by Merck Group (2 mentions)
Lsm 780 confocal microscope, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Quartz microtube, by Hellma (2 mentions)
D fl epi fluorescence attachment, by Nikon (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Dm2500 led, by Leica camera (2 mentions)
Cellometer counting chamber, by Revvity (2 mentions)
Eclipse 50i microscope, by Nikon (1 mentions)
Cell laboratory quantasctm cytometer, by Beckman Coulter (1 mentions)
Pna fitc, by Vector Laboratories (1 mentions)
126 protocols using live dead sperm viability kit
1
Assessing Sperm Viability with SYBR-14 and PI Staining
2
Sperm Membrane Integrity Evaluation
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The specimens were stained using the Live/Dead Sperm Viability Kit (Molecular Probes Inc., Leiden, The Netherlands). A 5 μL volume of 50× diluted SYBR-14 was added to 1 mL of diluted ejaculate, followed by incubation at 36 °C for 10 min. Then 5 μL of propidium iodide (PI) was added, followed by incubation at 36 °C for 10 min. A drop of solution was applied to a heated microscope slide, and the integrity of the cell membranes was examined using a Nikon Eclipse 50i microscope with a fluorescence. One slide per sample were analyzed. On each slide, 200 sperm were evaluated. Sperm emitting green fluorescence over the entire head were identified as live cells (with an intact cell membrane stained by SYBR-14), sperm emitting red fluorescence over the entire head or on part of the head and sperm emitting yellow-orange fluorescence over the entire head were identified as dead (with a damaged cell membrane, stained by PI), and sperm emitting yellow-orange fluorescence over the entire head were identified as moribund sperm.
3
Sperm Viability Evaluation via Flow Cytometry
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Sperm viability was evaluated through the assessment of membrane integrity using the LIVE/DEAD Sperm Viability Kit (Molecular Probes, Eugene, OR, United States), following the protocol of Garner and Johnson (1995) (link). Briefly, sperm (1 × 106 spermatozoa/mL) were incubated with SYBR-14 (final concentration: 100 nmol/L) for 10 min and then with Propidium Iodide (final concentration: 12 μmol/L) for 5 min at 38°C. Samples were evaluated using a Cell Laboratory QuantaSCTM cytometer (Beckman Coulter; Fullerton, CA, United States) and were excited with an argon ion laser (488 nm) set at a power of 22 mW.
In flow cytometry dot-plots, three sperm populations were observed: (1) viable, green-stained sperm (SYBR-14+/PI–); (2) non-viable, red-stained sperm (SYBR-14–/PI+); (3) non-viable, both red- and green-stained sperm (SYBR-14+/PI+). The fourth dot population corresponded to unstained, non-sperm particles (SYBR-14–/PI–). Viable green-stained sperm were used to assess sperm viability, and SYBR-14 fluorescence spill over into FL3-channel was compensated (2.45%).
In flow cytometry dot-plots, three sperm populations were observed: (1) viable, green-stained sperm (SYBR-14+/PI–); (2) non-viable, red-stained sperm (SYBR-14–/PI+); (3) non-viable, both red- and green-stained sperm (SYBR-14+/PI+). The fourth dot population corresponded to unstained, non-sperm particles (SYBR-14–/PI–). Viable green-stained sperm were used to assess sperm viability, and SYBR-14 fluorescence spill over into FL3-channel was compensated (2.45%).
4
Evaluating Sperm Plasma Membrane Integrity
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Plasma membrane integrity was evaluated using the LIVE/DEAD® sperm viability kit (Molecular Probes, Thermo Fisher Scientific; Waltham, MA, USA). Briefly, spermatozoa, previously diluted to a final concentration of 1 × 106 sperm/mL, were stained with SYBR14 (final concentration: 100 µM) for 10 min at 38 °C in the dark. Following this, spermatozoa were incubated with propidium iodide (PI; final concentration: 12 µM) for 5 min at the aforementioned conditions. Fluorescence of SYBR14 was detected through FL1, whereas that of PI was collected through FL3. Each spermatozoon was classified as with an intact (SYBR14+/PI−, green) or non-intact plasma membrane (SYBR14+/PI+, orange; or SYBR14−/PI+, red). Unstained and single-stained samples were used for setting the EV-gain, and FL1 and FL3 PMT voltages. Spillover from FL1 into the FL3 channel was compensated (2.45%). Percentages of non-sperm, debris particles appearing in the lower left quadrant (SYBR14−/PI−) were used to correct percentages of particles corresponding to sperm in this and other tests.
5
Sperm Membrane Integrity Assessment
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Sperm membrane integrity was determined using the LIVE/DEAD® Sperm Viability Kit (Molecular Probes, ThermoFisher Scientific; Waltham, MA, USA). This kit employs a mixture of two dyes: 1) SYBR-14, a membrane permeable fluorochrome that stains the sperm head in green (viable spermatozoa); and 2) propidium iodide (PI), a membrane impermeable fluorochrome that only penetrates through disrupted plasma membranes, staining sperm heads in red (non-viable spermatozoa). Briefly, samples were first incubated with SYBR-14 (final concentration: 100 nM) for 10 min at 37.5 °C in darkness, and then with PI (final concentration: 12 µM) for 5 min at 37.5 °C in the dark. Stained samples were evaluated with the flow cytometer, and only those spermatozoa showing positive SYBR-14 staining and negative PI staining (SYBR-14+/PI−) were considered as viable, i.e., membrane intact sperm. Results are expressed as mean ± SEM (n = 10) (Figure 6 ).
6
Evaluating Frozen-Thawed Microminipig Sperm
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The motility and quality of frozen–thawed spermatozoa from three
microminipig boars and a Large White boar were examined. Briefly, motility
analyses of frozen–thawed spermatozoa were performed using the
computer-assisted sperm analysis system (sperm class analyser: SCA v.4.2;
MICROPTIC, Barcelona, Spain). The motility analysis was based on the
examination of 25 consecutive digitized images obtained from three to five fields
using a 10 phase contrast objective, and at least 500 spermatozoa
per sample were analysed using an image capture speed of 40 ms.
Viability, plasma membrane integrity, and acrosome integrity analyses were
conducted using a live–dead stain combination (SYBR-14/propidium iodide
(PI), LIVE/DEAD Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR,
USA), the hypo-osmotic swelling test (Ahmad et al., 2003), and fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA; Vector
Laboratories, Inc., Burlingame, CA, USA), respectively, according to the
methods described by Wittayarat et al. (2012).
microminipig boars and a Large White boar were examined. Briefly, motility
analyses of frozen–thawed spermatozoa were performed using the
computer-assisted sperm analysis system (sperm class analyser: SCA v.4.2;
MICROPTIC, Barcelona, Spain). The motility analysis was based on the
examination of 25 consecutive digitized images obtained from three to five fields
using a 10 phase contrast objective, and at least 500 spermatozoa
per sample were analysed using an image capture speed of 40 ms.
Viability, plasma membrane integrity, and acrosome integrity analyses were
conducted using a live–dead stain combination (SYBR-14/propidium iodide
(PI), LIVE/DEAD Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR,
USA), the hypo-osmotic swelling test (Ahmad et al., 2003), and fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA; Vector
Laboratories, Inc., Burlingame, CA, USA), respectively, according to the
methods described by Wittayarat et al. (2012).
7
Sperm Viability Assessment using LIVE/DEAD Kit
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Sperm viability was evaluated by assessing plasma membrane integrity with LIVE/DEAD Sperm Viability Kit (Molecular Probes, Eugene, OR, USA). Sperm diluted at a final concentration of 1 × 106 in pre-warmed PBS were first incubated with SYBR14, a membrane-permeable fluorochrome that stains sperm heads in green, for 10 min (final concentration: 32 nmol/L). Thereafter, samples were incubated with propidium iodide (PI), a membrane-impermeable fluorochrome that only penetrates membrane-damaged sperm, for 5 min (final concentration: 7.5 μmol/L).
Stained sperm were examined through a CytoFLEX cytometer (Beckman Coulter; Fullerton, CA, USA). Samples were excited with an argon-ion laser at 488 nm and 10,000 events per replicate were evaluated, using FITC (BP 525/40) and PC5.5 (690/50) filters for SYBR14 and PI, respectively. Three separate populations were identified, a membrane-intact sperm population (SYBR14+/PI−) and two subpopulations of membrane-damaged sperm with different degrees of alteration (SYBR14+/PI+ and SYBR14−/PI+). Three technical replicates per sample were examined, and data were not compensated.
Stained sperm were examined through a CytoFLEX cytometer (Beckman Coulter; Fullerton, CA, USA). Samples were excited with an argon-ion laser at 488 nm and 10,000 events per replicate were evaluated, using FITC (BP 525/40) and PC5.5 (690/50) filters for SYBR14 and PI, respectively. Three separate populations were identified, a membrane-intact sperm population (SYBR14+/PI−) and two subpopulations of membrane-damaged sperm with different degrees of alteration (SYBR14+/PI+ and SYBR14−/PI+). Three technical replicates per sample were examined, and data were not compensated.
8
Acrosome and Membrane Integrity of Thawed Sperm
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Plasma membrane integrity and the acrosomal status of frozen-thawed spermatozoa were assessed by propidium iodide (PI) and peanut agglutinin (PNA) staining, respectively, as previously described [30 (link)]. Briefly, after preincubation of thawed spermatozoa for 1 h, a 20 μl aliquot of suspension was placed in a tube and stained with 1 μl of lectin peanut agglutinin (PNA) conjugated with Alexa Fluor
488 (1 mg/ml, Invitrogen, Paisley, UK) mixed with 0.5 μl of PI (2.4 mM, LIVE/DEAD® Sperm Viability Kit; Molecular Probes, Eugene, OR, USA) without fixation. The tube was incubated in a dark box at 37°C for 15
min. After incubation, 100 spermatozoa per sample were observed under a fluorescent microscope and classified into three staining patterns: PI(+), PI(–)/PNA(–), and PI(–)/PNA(+). PI(+) represents dead spermatozoa and
PI(–)/PNA(–) represents live spermatozoa with an intact acrosome. PI(–)/PNA(+) represents live spermatozoa ready to undergo the acrosome reaction; this state allows PNA to reach the acrosomal contents through pores in
the outer acrosomal membrane and plasmalemma.
488 (1 mg/ml, Invitrogen, Paisley, UK) mixed with 0.5 μl of PI (2.4 mM, LIVE/DEAD® Sperm Viability Kit; Molecular Probes, Eugene, OR, USA) without fixation. The tube was incubated in a dark box at 37°C for 15
min. After incubation, 100 spermatozoa per sample were observed under a fluorescent microscope and classified into three staining patterns: PI(+), PI(–)/PNA(–), and PI(–)/PNA(+). PI(+) represents dead spermatozoa and
PI(–)/PNA(–) represents live spermatozoa with an intact acrosome. PI(–)/PNA(+) represents live spermatozoa ready to undergo the acrosome reaction; this state allows PNA to reach the acrosomal contents through pores in
the outer acrosomal membrane and plasmalemma.
9
Sperm Membrane Integrity Assessment
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Sperm membrane integrity (SMI) was assessed using the two fluorescent dyes SYBR-14/propidium iodide (LIVE/DEAD Sperm Viability Kit, Molecular Probes®, Invitrogen, Carlsbad, CA, USA), as described by Rosato and Iaffaldano [32 (link)], but with minor modifications [31 (link),33 (link)]. Sperm cells (n = 200) were evaluated in duplicate aliquots for every sample using a Zeiss (Axioskop 40-AxioCamICc 1) microscope and FITC filter fluorescence at 100× total magnification.
10
Fluorescent Labeling of Live and Dead Sperm
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