The overall goal of this procedure is to obtain image series from immobilized human sperm labeled with a fluorescent calcium reporter die First. Motil cells are isolated from seminal plasma by swimming them up into incubation medium. After capacitation cells are labeled with fluorescent calcium indicator, then transferred to a fusible imaging chamber, which is mounted on an inverted fluorescence microscope.
Calcium ion concentration responses are captured as a time-lapse image series and analyzed to extract single cell responses. Hi, I'm Edwin Andres Morales Garcia from the laboratory of Dr.Steven J.Public Cova in the School of Biosciences at the University of Birmingham uk. I'm Katherine Nash, also from the Public Overlap.
And I'm Jennifer Morris from the Public Overlap. Today we'll show your procedure for imaging intracellular calcium responses in individual human perato soil. We use this procedure within our laboratory to study regulation of sperm activities using stimuli derived from the egg and the female trap.
So let's get started. Store semen samples at 37 degrees Celsius for no more than 30 minutes from the moment of collection to isolate motor cells from the seminal plasma PET one milliliter of L'S balance salt solutions supplemented with 0.3%BSA, abbreviated as S-E-B-S-S into a series of five milliliter polystyrene round bottom tubes. Gently underlay the S-E-B-S-S in each tube with 0.3 milliliters of semen and cap the tube loosely.
Incubate the tubes at a 45 degree angle for one hour at 37 degrees Celsius and 6%carbon dioxide. To allow motile cells to swim up into the S-E-B-S-S gently pip us off the top 0.7 milliliter of solution and pull the solution from all tubes into a clean 15 milliliter polystyrene centrifuge tube. Shake gently to mix count cells that have been immobilized with formalin in a new bowel counting chamber.
Bring the concentration to 6 million cells per milliliter in S-E-B-S-S and divide the sperm into 200 microliter aliquots in five milliliter polystyrene round bottom tubes. Cap loosely. If the response of capacitated cells is being studied, incubate the tubes for five to six hours at 37 degrees Celsius and 6%carbon dioxide to allow capacitation Apply 10 microliters of 10%polylysine as a number of small drops to the center of a 22 by 50 millimeter.
Number one cover slip and allow it to dry completely. Use vacuum grease to attach the cover slip treated side up to a fusible imaging chamber built. For the purpose.
For each imaging experiment, select a single tube of capacitated cells and add 1.2 microliters of 2.5 milligrams per milliliter. Calcium dependent organ green dissolved in DMSO for a final concentration of 10 micromolar. Incubate the tube for 40 minutes at 37 degrees Celsius and 6%carbon dioxide.
Using a 200 microliter Pepto with a P 200 tip, remove all of the solution from the tube completely. Fill the chamber by gently injecting into the inflow port. Tilt the chamber so that bubbles escape through the outflow.
Place the entire chamber into the incubator and incubate for a further 20 minutes at 37 degrees Celsius and 6%carbon dioxide. To allow the sperm to adhere to the poly D lysine. Mount the chamber to the inverted fluorescent microscope and connect it to the perfusion apparatus.
Leave the preparation in the dark for at least 10 minutes to let the perfusion sweep away. Loose cells and extracellular die under phase contrast at 40 x or 60 x magnification, choose a field of view where cell spacing is sufficient to permit fluorescence from each cell to be assessed separately. Cells should be adequately attached, but preferably exhibiting clear fledger activity.
Switch the microscope to the green fluorescent channel. Keep exposure times at the minimum required for a clear fluorescent image. To reduce photobleaching in IQ software.
Activate the time series image acquisition. Acquire images at 0.1 hertz and use a software controlled shutter to limit illumination. Initially recording for a control period of three to five minutes, manipulate the saline's calcium concentration and apply drugs by changing saline at the header of the perfusion system.
Note to the time that each stimulus is added to the header and calculate its arrival time in the chamber as described in the written portion of this protocol. To analyze images offline, use one of the drawing tools to outline a region of interest or ROI around each cell or part of a cell here, the posterior sperm head because stored calcium as mobilized in this area. Scroll with the slider bar to check the image series.
Identify and remove cells that fail to remain fully within the ROI overlap other cells or have shown the large and rapid increase followed by sharp drop-off of fluorescence intensity characteristic of cell death. Use the analyze function to generate a time fluorescence intensity series for each ROI. The data can then be exported and analyzed in a spreadsheet sheet.
These images are from a series obtained during stimulation with three micromolar progesterone. The lower row has been coated in pseudo color. To better illustrate relative intensity changes, progesterone entered the chamber here.Here.
These results are animated in a time-lapse video. Notice the rapid spike in fluorescence intensity followed by a slow increase indicating corresponding changes in intracellular calcium levels. These data are quantified here from average intensities of the posterior head of three cells.
Three micromolar progesterone was added at the time marked by the arrow. The dash line shows mean control fluorescence. Today we just shown you how to isolate good quality cells, load them with a calcium sensitive dye, and monitor the response of individual cell to stimulation.
When doing this procedure, it's important to remember to use the best possible cell population, also minimize illumination of the cells to avoid photo damage and pick the dye that suits the responses you're looking at best. So that's it. Thanks for watching and good luck with your experiments.
