Name: Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging (Video) | JoVE Journal
Uploaded: 2013-05-24T12:00:00.000+00:00
Duration: 19 min 26 s
Description: Learn to measure intracellular calcium fluctuations in human sperm using four fluorescent techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry, and single cell imaging.

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The authors thank Jose Luis De la Vega, Erika Melchy and Dr. Takuya Nishigaki for technical assistance. This work was supported by Consejo Nacional de Ciencia y Tecnolog&iacute;a (CONACyT-Mexico) (99333 and 128566 to CT); Direcci&oacute;n General de Asuntos del Personal Acad&eacute;mico/ Universidad Nacional Aut&oacute;noma de M&eacute;xico (IN202212-3 to CT).

<ol><li>Bouschet, T., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+as+an+extracellular+signalling+molecule%3A+perspectives+on+the+Calcium+Sensing+Receptor+in+the+brain%5BTitle%5D)+AND+%22Comptes+Rendus+Biologies%22%5BJournal%5D)">Calcium as an extracellular signalling molecule: perspectives on the Calcium Sensing Receptor in the brain.</a> Comptes Rendus Biologies. 328, 691-700 (2005).</li>
<li>Darszon, A., Nishigaki, T., Beltran, C., Trevino, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+channels+in+the+development%2C+maturation%2C+and+function+of+spermatozoa%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">Calcium channels in the development, maturation, and function of spermatozoa.</a> Physiol. Rev. 91, 1305-1355 (2011).</li>
<li>Esposito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mice+deficient+for+soluble+adenylyl+cyclase+are+infertile+because+of+a+severe+sperm-motility+defect%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 2993-2998 (2004).</li>
<li>Avenarius, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+male+infertility+caused+by+mutations+in+the+CATSPER1+channel+protein%5BTitle%5D)+AND+%22American+Journal+of+Human+Genetics%22%5BJournal%5D)">Human male infertility caused by mutations in the CATSPER1 channel protein.</a> American Journal of Human Genetics. 84, 505-510 (2009).</li>
<li>Carlson, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+targeting+of+native+CatSper+channels+reveals+a+required+role+in+maintenance+of+sperm+hyperactivation%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Pharmacological targeting of native CatSper channels reveals a required role in maintenance of sperm hyperactivation.</a> PLoS ONE. 4, e6844(2009).</li>
<li>Brokaw, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+and+flagellar+response+during+the+chemotaxis+of+bracken+spermatozoids%5BTitle%5D)+AND+%22J.+Cell.+Physiol%22%5BJournal%5D)">Calcium and flagellar response during the chemotaxis of bracken spermatozoids.</a> J. Cell. Physiol. 83, 151-158 (1974).</li>
<li>Visconti, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacitation+of+mouse+spermatozoa.+II.+Protein+tyrosine+phosphorylation+and+capacitation+are+regulated+by+a+cAMP-dependent+pathway%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.</a> Development. 121, 1139-1150 (1995).</li>
<li>Svahn, H. A., van den Berg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cells+or+large+populations%5BTitle%5D)+AND+%22Lab+on+a+chip%22%5BJournal%5D)">Single cells or large populations.</a> Lab on a chip. 7, 544-546 (2007).</li>
<li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+fluorescence+microscopy+for+systems+biology%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">High-throughput fluorescence microscopy for systems biology.</a> Nat. Rev. Mol. Cell Biol. 7, 690-696 (2006).</li>
<li>Strunker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CatSper+channel+mediates+progesterone-induced+Ca2%2B+influx+in+human+sperm%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm.</a> Nature. 471, 382-386 (2011).</li>
<li>Lishko, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Control+of+Male+Fertility+by+Spermatozoan+Ion+Channels%5BTitle%5D)+AND+%22Annu.+Rev.+Physiol%22%5BJournal%5D)">The Control of Male Fertility by Spermatozoan Ion Channels.</a> Annu. Rev. Physiol. , (2011).</li>
<li>Kao, J. P., Harootunian, A. T., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photochemically+generated+cytosolic+calcium+pulses+and+their+detection+by+fluo-3%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Photochemically generated cytosolic calcium pulses and their detection by fluo-3.</a> J. Biol. Chem. 264, 8179-8184 (1989).</li>
<li>Kilic, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caged+progesterone%3A+a+new+tool+for+studying+rapid+nongenomic+actions+of+progesterone%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Caged progesterone: a new tool for studying rapid nongenomic actions of progesterone.</a> Journal of the American Chemical Society. 131, 4027-4030 (2009).</li>
<li>Lishko, P. V., Botchkina, I. L., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progesterone+activates+the+principal+Ca2%2B+channel+of+human+sperm%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Progesterone activates the principal Ca2+ channel of human sperm.</a> Nature. 471, 387-391 (2011).</li>
<li>Xia, J., Ren, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+BSA-induced+Ca2%2B+influx+during+sperm+capacitation+is+CATSPER+channel-dependent%5BTitle%5D)+AND+%22Reprod.+Biol.+Endocrinol%22%5BJournal%5D)">The BSA-induced Ca2+ influx during sperm capacitation is CATSPER channel-dependent.</a> Reprod. Biol. Endocrinol. 7, 119(2009).</li>
<li>Galantino-Homer, H. L., Florman, H. M., Storey, B. T., Dobrinski, I., Kopf, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine+sperm+capacitation%3A+assessment+of+phosphodiesterase+activity+and+intracellular+alkalinization+on+capacitation-associated+protein+tyrosine+phosphorylation%5BTitle%5D)+AND+%22Mol.+Reprod.+Dev%22%5BJournal%5D)">Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation.</a> Mol. Reprod. Dev. 67, 487-500 (2004).</li>
<li>Baldi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+calcium+accumulation+and+responsiveness+to+progesterone+in+capacitating+human+spermatozoa%5BTitle%5D)+AND+%22J.+Androl%22%5BJournal%5D)">Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.</a> J. Androl. 12, 323-330 (1991).</li>
</ol>

We have nothing to disclose.

Intracellular signaling is vital for most cellular activities; Ca2+ is a ubiquitous messenger that accompanies mammalian cells throughout their entire lifespan, from their origin at fertilization, to the end of their life cycle. In response to different stimuli, [Ca2+]i increases, oscillates and decreases with spatio-temporal codification; accordingly, diverse processes are activated, modulated or terminated by Ca2+-encoded messages. Intracellular Ca2+ dynamics are very ...

Ca2+ is a universal second messenger of signal transduction pathways in eukaryotic cells. Intracellular Ca2+ (Ca2+i) participates in the regulation of many fundamental physiological processes in both excitable and non-excitable cells. The importance and universality of Ca2+ as second messenger during signal transduction events is derived from its spatio-temporal versatility in the transmission of information within the cell. While Ca2+ cannot be synthesized de novo or degraded within the cell, its intracellular concentration ([Ca2+]i) is maintained within very strict limits through different cellular mechanisms that continuously buffer, sequester, compartmentalize, and/or accumulate Ca2+. Changes in the concentration of this ion can occur in highly localized areas within the cell 1, and deciphering such fluctuations is essential for gaining a deeper understanding of (1) their role in the signaling mechanism, (2) their physiological significance, and (3) general mechanisms of cell signaling. Ca2+-mediated signaling is of particular importance in sperm physiology 2. Sperm motility is one of the most important functions for fertilization success, and in fact, several sperm motility defects can cause sterility 3-5. The importance of Ca2+ in flagellar movement has been long recognized 6; however, the mechanism of how Ca2+ controls the specific form of flagellar bending is not fully understood.
Before fusing with the egg, spermatozoa must undergo capacitation, a complex process dependent on sperm residence inside the female tract. During capacitation, the sperm membrane's lipid architecture and organization are modified, mainly as a result of cholesterol removal from the plasma membrane. Additionally, several proteins are tyrosine-phosphorylated 7. Importantly, during capacitation there is an increase in intracellular pH (pHi) and in [Ca2+]i, and the membrane potential hyperpolarizes in some species 2. Capacitation only takes place in a subpopulation of spermatozoa (20-40%), and the mechanisms involved in all these cellular changes are far from clear. It is generally accepted that only a subpopulation of capacitated sperm undergo the acrosome reaction (AR) when exposed to physiological inductors. The AR is also a Ca2+-regulated event required for fertilization in all species possessing an acrosome (specialized organelle with outer and inner membranes). During this process the outer acrosomal membrane fuses with the sperm's plasma membrane, releasing hydrolytic enzymes that allow the sperm cell to penetrate the glyco-proteinaceous matrix surrounding the egg (zona pellucida, or ZP). The AR also exposes a new fusogenic sperm cell surface that interacts with the egg plasma membrane for the final fusion of both gametes. There are several cellular ligands that induce the AR, progesterone being one of the most studied among them.
In this work we present four different techniques involving the use of a Ca2+-sensitive fluorescent dye to measure [Ca2+]i changes in human sperm triggered by progesterone (except for flow cytometry, in which we measured the [Ca2+]i increase induced during the in vitro capacitation process). In this particular case we used Fluo-3 AM (Life Technologies, Grand Island, NY), a membrane-permeable dye with a Kd = 325 nM. In vitro we monitored fluorescence changes as a function of time with three of the methodologies, and with the fourth technique we measured fluorescence values at a single given point in time. These different approaches complement each other, since altogether they provide spatial and temporal resolution at both the single cell and cell population levels.
Cell Population or Bulk Experiments
Bulk techniques are extensively used not only because the instruments they require are readily available, but also because they are simple, well established, and allow for the averaging of information from measurements performed on millions of cells in a single experiment.
Technique #1. Conventional Fluorometry 
This technique monitors changes in fluorescence as a function of time; the experiments are performed in glass cuvettes with sample volumes ranging from 200 to 1,000 &mu;l. Proper mixing of added reagents requires magnetic stirring, and therefore the temporal resolution obtained is in the order of seconds. The typical cell concentration range of the samples analyzed is 105-108 cells/ml.
Technique #2. Stopped Flow Fluorometry 
This technique also monitors changes in fluorescence as a function of time, but the reagents are rapidly mixed together (using pressure) into a recording cuvette containing a very small sample volume (ranging from 25-100 &mu;l). Therefore, homogenization of reagents is instantaneous, enabling a high temporal resolution in the order of milliseconds. Analysis of the resulting fluorescence vs. time traces are suitable for determining reaction rates, elucidating the complexity of the reaction mechanism, obtaining information on short-lived reaction intermediates, etc. The common cell concentration range of the samples analyzed is 105-107 cells/ml.
Single Cell Experiments
Bulk experiments report the average behavior of a large number of cells; however, a population may frequently exhibit heterogeneous properties that are overlooked during such type of measurements. Single cell techniques are thus used to complement the information obtained with cell population experiments.
Technique #3. Flow Cytometry 
Despite the importance of the information arising from single cell measurements, it is important to analyze a large number of cells in order to prevent the erroneous extrapolation of cell-specific properties to an entire population. For this reason, high-throughput techniques are favored and the most popular method is flow cytometry, in which 10,000 cells per condition are conventionally analyzed. This method enables multi-parametric analysis of heterogeneous populations as it categorizes cells according to their size (forward scatter (FSC)), granularity (side scatter (SSC)) and fluorescence intensity (specific labeling with an antibody, viability marker, etc.), thus providing information on the parameters' distribution for a group of cells. Flow cytometry provides instant rather than time-dependent information 8. Forward and side scatter values are also useful for selecting a gate that includes cells but discriminates cellular debris, dust, etc. For fluorescence measurements, negative and positive fluorescence controls must also be included. If more than one fluorescence channel is used, a process known as compensation must be performed (for details see <a href="http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp" target="_blank">http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp</a>). Compensation allows for spectral overlap discrimination among fluorophores. Flow cytometry also allows discrimination of dead cells, generally by means of propidium iodide staining.
Technique #4. Single Cell Imaging 
Microscopy is another common method to study single cell behavior; it is well suited for time-dependent studies and it also provides spatial resolution. A major drawback is that high-throughput analysis is only in its infancy at the present time 9.

<table><tbody><tr><td>Ham&#039;s F-10</td><td>Sigma-Aldrich</td><td>N-6013</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A-7906</td><td></td></tr><tr><td>Calcium Chloride Dihydrate approx. 99%</td><td>Sigma-Aldrich</td><td>C-3881</td><td></td></tr><tr><td>Makler Counting Chamber</td><td>SEFI Medical Insruments LTD</td><td>SEF-MAKL</td><td></td></tr><tr><td>Fluo-3 AM</td><td>Invitrogen</td><td>F-1242</td><td>20 vials/50 &amp;mu;g each</td></tr><tr><td>Ionomycin</td><td>Alomone</td><td>I-700</td><td></td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P0130</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S-9888</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P-3911</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Sodium bicarbonate</td><td>JT Baker</td><td>3506</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M-2670</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Calcium chloride anhydrous</td><td>Sigma-Aldrich</td><td>C-1016</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-3125</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>D-Glucose</td><td>JT Baker</td><td>1906-01</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P-2256</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Sodium L-lactate (aprox. 99%)</td><td>Sigma-Aldrich</td><td>L- 7022</td><td>Reagents for human sperm medium (HSM)</td></tr><tr><td>Propidium Iodide </td><td>Invitrogen</td><td>L-7011</td><td>Component B</td></tr><tr><td>Triton X-100 (t-Octylphenoxypolyethoxyethanol)</td><td>Sigma- Aldrich</td><td>X-100</td><td>2.4 mM solution in water </td></tr><tr><td>Round coverslip </td><td>VWR</td><td>48380 080</td><td>25 mm diameter</td></tr><tr><td>Poly-L-lysine solution </td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma-Aldrich</td><td>M-3634</td><td></td></tr><tr><td>Attofluor; Cell Chamber, for microscopy</td><td>Life technologies</td><td>A-7816</td><td></td></tr><tr><td>Dimethyl Sulphoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td>5x5 ml</td></tr></tbody></table>

measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

<img alt="Semen capacitation process diagram; includes sample preparation, incubation, cell counting with grid." fo:content-width="6in" fo:src="/files/ftp_upload/50344/50344fig1highres.jpg" src="/files/ftp_upload/50344/50344fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. Schematic diagram of the experimental protocol for sperm sample preparation by the swim-up method. The major steps for separation of motile sperm and for adjustment of...

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

The overall goal of this procedure is to measure intracellular calcium fluctuations in human sperm with fluorescent dyes. This is accomplished by first preparing the sperm sample by the swim up method and if needed, promoting capacitation. The second step is to load the sperm with a calcium fluorescent dye.Next, using conventional fluorometry stopped flow fluorometry, flow cytometry or single cell imaging, perform the experiment and record the calcium measurements. Ultimately, the results are analyzed to determine changes in intracellular calcium concentrations triggered by progesterone or during the capacitation process. The main advantage of this technique over existing methods like those involving radioactivity is that this is a very sensitive procedure.It is safe and easy to perform. This method can help answer key questions in the SPR calcium signaling field, such as identification of compounds that induce intracellular calcium, increases with high spatial and temporal resolution and identification of different cellular sub populations in a siemen sample. The implications of using, for example, the flow cytometry technique extend towards diagnosis of fertility problems through the analysis of the physiological state of male gamuts.Even though this method can provide insights into the study of calcium mobilization of human sperm, it can also be applied to other type of cells such as male gamuts from other species or different type of cells such as neurons or muscle cells. Generally, individuals who use this method will struggle because the sperm handling is not easy and it is important to maintain appropriate conditions to obtain viable and motor cells through the experiment. We implemented the use of these techniques by combining strategies using different fields.Visual demonstration of this method is important as preparation and handling of the sample, as well as the addition of the compounds are difficult to learn because sperm cells can be damaged during the procedure and artifacts in the responses could be obtained during the edition of the compounds. To prepare the sperm sample, one milliliter of hams F 10 medium must be carefully layered on top of each 500 microliter liquified semen. Eloqua, touch the wall of the tube with the tip of the micro pipet and gently dispense the medium above the sample.It is crucial to do it slowly to avoid mixing the sample and medium layers. The medium is supplemented with two millimolar calcium chloride and five milligrams per milliliter of BSA. To promote capacitation in vitro, carefully lean the tubes to approximately a 30 degree angle.This will increase the surface area between the two liquids, thus enhancing the displacement or swim up of sperm cells from the sample to the medium during incubation. Next, place the lean test tubes inside an incubator at 37 degrees Celsius and 5%carbon dioxide. 95%air for one hour after one hour.Use a micro pipet to carefully remove from each tube the upper 700 microliters of the hams F 10 medium that now contains motil spermatozoa. Pool all the collected samples into a single clean glass tube, avoiding bubble formation. Place 10 microliters of pooled sample on the optical flat glass of a macular counting chamber base and place the cover glass.Make sure to avoid bubble formation inside the chamber as this would result in an inaccurate cell count. Observe under a compound microscope equipped with a 20 x objective. The cover glass of the macular counting chamber has a big square composed of 100 smaller squares.Count the cells in any strip of 10 squares. This number represents the cell concentration in millions of cells per milliliter. Repeat the count in two additional 10 square strips and calculate the mean of the three counts to load cells with a fluorescent dye.Combine in a 1.5 milliliter fuge tube, the required volume of sperm suspension with enough one millimolar flu oh 3:00 AM stock solution to obtain a final concentration of two micromolar flu oh 3:00 AM incubate for 30 minutes at 37 degrees Celsius and protected from light centrifuge the tube at 750 G for five minutes. The formation of a cloud rather than a pellet indicates that the cells are in good condition, aspirate and discard the supernatant and resuspend the pellet in the appropriate volume of human sperm medium or HSM. To begin this experiment, combine in a flat bottom glass tube, 570 microliters of HSM and 30 microliters of sperm cell suspension previously loaded with flu oh 3:00 AM and resuspended in HSM to obtain one times 10 to the eighth cells per milliliter.Place a magnetic stir bar inside the glass tube and insert the tube into the reading chamber of the spectometer preheated to 37 degrees Celsius. The sample must be stirred throughout the acquisition time. Start the experiment using the oli software and proceed to acquire fluorescence values at a frequency of 0.5 hertz during 300 seconds.After acquiring basal fluorescence for 30 seconds, use a Hamilton Micro syringe to inject the appropriate volume of test compound in this case for micromolar progesterone at 100 seconds. At 20 micromolar ion mycin as a positive control to obtain the maximum fluorescence value. In this experiment, intracellular calcium changes are measured with high temporal resolution.Using an SFM 20 stopped flow mixer coupled to a rabid kinetics optical system, fill one of the instrument syringes with one milliliter of flu. 3:00 AM loaded sperm cells and the second syringe with one milliliter of the compound to be tested. 20 micromolar progesterone dissolved in HSM.In this example, it is crucial to avoid bubble formation while drawing the liquids into the syringes. Lift both instrument pistons until they touch the tip of the syringe plungers. Set the total sampling time in this case to 50 seconds and the frequency to 10 milliseconds in order to minimize cell damage, set the flow rate to the minimum value that will provide a measurable response trigger.The mixing of reagents, the trace of raw fluorescence versus thyme will be displayed on the computer screen. Repeat this procedure replacing the progesterone with HSM as a negative control and 10 micromolar ion mycin as a positive control prior to flow cytometry. Prepare the experimental samples by placing 500 microliters of cell suspension per cytometer tube under each condition to be tested.Set up an experiment using the equipment software. First, create a new folder, experiment specimen, and number of tubes. Then select appropriate cytometer settings for flu O 3:00 AM.Use the fluorescein isothiocyanate and propidium iodide filters.Run unstained control tubes one and two in the cytometer. Collect forward and side scatter data to verify that threshold settings are appropriate and to create the corresponding gate in order to discriminate debris from cells. Run the experimental tubes and collect fluorescence data from 10, 000 events per sample.At the end. Export all data to the software available for analysis. Prepare round cover slips needed for this procedure by applying a five microliter drop of poly L lysine solution onto the center of each cover slip.Leave for at least one hour prior to use rinse treated area with water. This will remove excess polylysine and allow sperm cells to adhere to the cover slip from their head while their flagella can still move. Assemble the cover slip inside the recording chamber.Place 10 microliters of flu oh 3:00 AM loaded cells at a concentration of one times 10 to the seventh cells per milliliter. In the center of the cover slip. Cover the cells with 200 microliters of pre prewarm HSM.Place the chamber on the stage of the microscope, preheated to 37 degrees Celsius, and view the cells using phase contrast. Select an area where cell density is appropriate for imaging. Too many cells make analysis difficult due to overlapping signals.Cells should be firmly attached to the cover slip by their head, but exhibit flagella movement, which confirms viability. Start the experiment by activating the time series image acquisition software. Typically four images are required per second with an illumination of two milliseconds per image.Acquire fluorescence images in live mode. To adjust the focus and brightness. Use a micro pipette to carefully add dropwise the test compound progesterone in this case and continue image acquisition as required.Perform two sequential control additions into the same chamber. 20 micromolar ion mycin to obtain maximal fluorescence and five millimolar manganese chloride to obtain minimal fluorescence. Perform image analysis offline using IQ software.Draw the regions of interest or ROIs around each cell or part of a cell. In addition, select a cell-free area for automatic background subtraction by the software. A time fluorescence intensity series is then obtained for each ROI and these data may be exported to Microsoft Excel for further analysis.Progesterone is one of the known acrosome reaction inducers and as expected provokes, a transient intracellular calcium concentration increase in human sperm as measured by conventional fluorometry. The red fluorescence trace versus time shows intracellular calcium concentration changes caused by the addition of four micromolar progesterone as a negative control HSM was added, which did not cause any change in intracellular calcium concentration levels as indicated by the blue fluorescence trace as a positive control. The change induced by 10 micromolar ion mycin is shown for each trace.Addition of this calcium ionophore causes the maximum RA cellular calcium concentration increase, which does not return to basal levels. This bar graph showed the average change in fluorescence delta F from each condition, plus or minus standard error where N equals three and the asterisk indicates a P value of less than 0.001 compared with the control. The progesterone induced intracellular calcium concentration increase was measured with greater temporal resolution by stopped flow fluorometry and representative results are shown here.Raw traces are shown in panel A, both progesterone represented by the red line and ion mycin represented by the blue line caused a rapid intracellular calcium concentration increase. The green trace is the negative control where cells were mixed with HSM. Panel B shows corrected traces for the signals induced with progesterone or with ion mycin obtained by subtracting the control signal from their corresponding raw signals.Progesterone caused a very fast and transient intracellular calcium concentration increase with a maximum fluorescence value occurring 2.7 seconds after addition of the inductor. On the other hand, CIN caused a rapid and sustained intracellular calcium concentration increase throughout 50 seconds. The inset in panel B shows an expanded view of the first 500 milliseconds for each response.The absence of a delay in the progesterone induced intracellular calcium concentration increase is consistent with previous reports suggesting that progesterone directly activates the calcium channel cat spur without intermediate signaling. Intracellular calcium concentration was also measured in capacitated and non capacitated human sperm using flow cytometry. In these representative forward and side scatter plots.Capacitated cells are in blue and non capacitated cells are in red. The selected gate is indicated with the blue line and only the cells within that area were used for further analysis. Panel B shows the fluorescence histogram in the Fitzy channel from Unstained, spermatozoa, and panel D shows the fluorescence histogram in the FZ channel from flu oh 3:00 AM stained spermatozoa as observed in Panel D.The distribution of fluorescence values for capacitated sperm represented by the blue trace is shifted to higher values compared to non capacitated sperm represented by the red trace.Panel C shows the fluorescence histogram in the PI channel from unstained cells and panel E shows the fluorescence histogram in the PI channel from dead cells. The fluorescence values for each individual cell can be observed in the two dimensional fluorescence dot plots shown here. For unstained cells and cells double stained with flu oh 3:00 AM and pi, the percentage of cells recorded in each quadrant is indicated by the red number.Importantly, the signal arising from dead cells can be eliminated. Lastly, the progesterone induced intracellular calcium concentration change was measured in single sperm cells by imaging these representative pseudocolor images show cells visualized at the beginning and after progesterone, CIN and manganese chloride additions. Progesterone addition causes an increment in intracellular calcium concentration, both in the sperm head and the lum manganese chloride is used to decrease the flu.Oh 3:00 AM fluorescence by quenching representative normalized fluorescence traces of nine individual cells from the experiment are shown in this figure. As observed in population experiments, single cell analysis revealed a transient and a sustained increase for progesterone and ion mycin respectively. Once mastered, this technique can be done in three to four hours if it is performed properly.While attempting this procedure, it is important to remember to verify the viability of the cells and to perform the appropriate controls Following this procedure. Other methods like electrophysiology could be performed in order to answer additional questions such as the identification of specific ion channels involved in the calcium increases by using a proppe solutions and simulation protocols After its development. This technique paved the way for researchers in the field of cell physiology to explore calcium dynamics in living cells with high spatial and temporal resolution.After watching this video, you should have a good understanding of how to select the most appropriate technique to measure intracellular calcium increases using fluorescent dy in any type of cell, depending on the specific question that you would like to answer. Don't forget that working with human siemen samples can be hazardous and precautions such as the use of donors of proven health. The use of latex globes during the procedure and appropriate disposal of solution and materials should always be taken while performing this procedure.

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Research

JoVE Journal

Biology

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Fluorimetric Techniques for the Assessment of Sperm Membranes

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Flow Cytometry Purification of Mouse Meiotic Cells

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide.  This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps.  We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Learn to measure intracellular calcium fluctuations in human sperm using four fluorescent techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry, and single cell imaging.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging (Video) | JoVE Journal

Measuring Intracellular Ca²⁺ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

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