Tissue homogenization, isolation of total cellular RNA, mRNA reverse transcription, and qPCR analysis were done as previously described [19 (link), 20 (link)]. Detection systems using fluorescent probes were purchased from Applied Biosystems. Expression values were normalized to abundance of mRNA transcripts of the house-keeping gene human hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT).
Fluorescent probes
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
Fluorescent probes are specialized laboratory equipment used for the detection and visualization of specific molecules or cellular structures. They consist of fluorescent dyes or molecules that emit light when excited by a specific wavelength of light. Fluorescent probes are designed to bind to or interact with target analytes, enabling the detection and quantification of these targets in various biological and chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Verteporfin, by Avantor (7 mentions)
Multiclamp 700b amplified, by Molecular Devices (2 mentions)
Taqman qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Fluoview fv1000mpe, by Olympus (1 mentions)
Bx61wi, by Olympus (1 mentions)
Facscanto 2 cytofluorometer, by BD (1 mentions)
Taqman microrna cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Isotype control, by Thermo Fisher Scientific (1 mentions)
Taqman quantitect probe pcr kit, by Qiagen (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Milli q, by Merck Group (1 mentions)
Ethanol, by Avantor (1 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
34 protocols using fluorescent probes
1
Quantitative RNA Expression Analysis
2
Quantitative Analysis of Inflammatory Markers
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RNA was extracted using RNeasy plus mini kit (Qiagen) and 1 μg total RNA reverse transcribed (Taqman Reverse Transcription kit; Applied Biosystems). Each reaction was performed with cDNA reverse transcribed from 10 ng RNA. qRT-PCR was performed using TaqMan Gene Expression Master Mix with TaqMan primers and fluorescent probes from Applied Biosystems for CXCL1 (Hs00605382_gl), DEFB4B (Hs00175474_ml), IL36G (Hs00219742_ml), IL36RN (Hs00202179_ml), LCN2 (Hs00194353_ml), MMP13 (Hs00233992_ml), PI3 (Hs00160066_ml), S100A7 (Hs00161488_ml), S100A8 (Hs00374264_gl), and S100A9 (Hs00610058_ml) on an Applied Biosystems Fast PCR machine. Target gene levels were normalized to the expression of the housekeeping gene RPLP0 (Hs99999902_ml).
3
Sperm Quality Analysis by Flow Cytometry
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The flow cytometric analysis was performed using an Acoustic Focusing Cytometer (Attune) equipped with blue (Argon 488 nm) and violet (UV 450 nm) lasers. The fluorescent probes were purchased from Invitrogen. The green, orange, and red fluorescence signals were detected using the photomultipliers BL1 (530/30 filter), BL2 (575/24 filter), and BL3 (>640 filter), respectively. The flower stability cytometer essence was evaluated daily using a standard solution (Invitrogen).
A total of 20,000 sperm events were examined per sample at a flow rate of 200 cells/s. In order to visualize the sperm population, the nonsperm events and cell debris were eliminated from the FSC × SSC scatter plots (4 ) through exposure to Hoescht 33342 (2 mM), followed by the detection of the fluorochrome using photomultiplier VL1 (filter 450/40); this process was not performed for the DNA fragmentation analysis. The results were obtained using the Attune Cytometric software V2.1.
A total of 20,000 sperm events were examined per sample at a flow rate of 200 cells/s. In order to visualize the sperm population, the nonsperm events and cell debris were eliminated from the FSC × SSC scatter plots (4 ) through exposure to Hoescht 33342 (2 mM), followed by the detection of the fluorochrome using photomultiplier VL1 (filter 450/40); this process was not performed for the DNA fragmentation analysis. The results were obtained using the Attune Cytometric software V2.1.
4
Characterization of Mouse Germ Cell Lines
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The chemicals and reagents used in this study were purchased from Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, USA) unless stated otherwise, and were of research grade. The fluorescent probes were purchased from Invitrogen (Carlsbad, CA, USA) unless otherwise stated. Mouse germ cell lines were purchased from the American Tissue Culture Collection (ATCC; Rockville, MD, USA). These cell lines included type B spermatogonia-like GC1 (ATCC CRL-2053) and primary spermatocyte-like GC2 (ATCC CRL-2196) strains. Human embryonic kidney (HEK) 293 (ATCC CRL-1573), McCoy mouse fibroblast (ATCC CRL-1696) and COV434 human granulosa (Sigma Aldrich) cell lines were also used for comparison.
5
Sperm Capacitation Assay Protocol
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All reagents and chemicals used in this study were of research grade and purchased from Merck, unless stated otherwise. Fluorescent probes were purchased from Invitrogen, unless stated otherwise. Freshly prepared Biggers, Whitten, and Whittingham (BWW) media (osmolarity of 290–310 mOsm/kg) (Biggers et al. 1971 ), was pre-warmed to 37°C and used for all sperm incubation experiments. When noncapacitated populations of spermatozoa were required, BWW medium was prepared with the omission of NaHCO3 (a strategy that prevents the initiation of capacitation-associated signaling cascades) but with the addition of 5.6 mM NaCl to maintain the osmolarity of the medium.
6
Multiphoton microscopy of cardiomyocytes
7
Whole-cell Recordings in CA3 Pyramidal Neurons
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Whole-cell recordings in CA3 pyramidal neurons were performed using 3-3.5 MΩ pipettes with an intracellular solution (in mM): K Gluconate 130, KCl 5, MgCl2 2, HEPES 10, di-tris-Phosphocreatine 10, NaATP 4, NaGTP 0.4 (pH adjusted to 7.2 with KOH, osmolarity 290-295 mOsM). The morphological tracer Alexa Flour 594 hydrazide (50μM) was also included in the intracellular solution. Voltage clamp recordings of miniature EPSCs were made, with the mEPSCs recorded at -70 mV. Receptor agonists and antagonists purchased from Tocris Cookson (Bristol, UK) were added to the aCSF at the following concentrations (in μM): TTX 1, Picrotoxin 100 and DCG-1V ((2S, 2′R, 3′R)-2-(2′, 3′-dicarboxycyclopropyl) glycine) 2. Recordings were performed with a Multiclamp 700B amplified (Molecular Devices). Recording sweeps were collected at 5kHz using WinWCP (Strathclyde Electrophysiology). mEPSCs were recorded over 15min periods in control and treated conditions, data were then analyzed off-line using Mini-Analysis software (Synaptosoft). mEPSCs were detected if their amplitude was greater than the threshold of 5pA, and considered for analysis if their rise time was shorter than their decay time. Fluorescentprobes were purchased from Invitrogen.
8
Whole-cell Recordings in CA3 Pyramidal Neurons
9
Fluorescent Probe Labeling Protocol
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Chemicals and reagents used throughout this study were purchased from Sigma-Aldrich (Sigma Chemical Co.) unless otherwise stated, and were of research grade. The fluorescent probes used were purchased from Invitrogen unless otherwise stated.
10
Cell Viability, Mitochondrial Mass, and Calcium Quantification
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Cell viability was evaluated via propidium iodide (PI) staining (5 μg/ml). Changes in mitochondrial mass were analyzed with dye Mitotracker green (100 nM, 30 min, 37°C). Cytosolic or mitochondrial Ca2+ quantifications were assessed with Fluo3-AM (2.5 μM, 30 min, RT) or with Rhod2-AM (2.5 μM, 30 min, 37°C). Fluorescences were analyzed on a FACSCanto II cytofluorometer (Beckton Dickinson). All Fluorescent probes were purchased from Life-Technologies.
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