Journal of Visualized Experiments www.jove.com Copyright © 2010 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License June 2010 | 40 | e1996 | Page 1 of 4 Video Article Techniques for Imaging Ca 2+ Signaling in Human Sperm Katherine Nash 1 , Linda Lefievre 2 , Ruben Peralta-Arias 1 , Jennifer Morris 1 , Aduen Morales-Garcia 1 , Tom Connolly 2 , Sarah Costello 1 , Jackson C. Kirkman-Brown 3 , Stephen J. Publicover 1 1 School of Biosciences, University of Birmingham 2 School of Medicine, University of Birmingham 3 Centre for Human Reproductive Science, Birmingham Women’s Hospital URL: https://www.jove.com/video/1996 DOI: doi:10.3791/1996 Keywords: Cellular Biology, Issue 40, sperm, human, calcium, fluorescence microscopy Date Published: 6/16/2010 Citation: Nash, K., Lefievre, L., Peralta-Arias, R., Morris, J., Morales-Garcia, A., Connolly, T., Costello, S., Kirkman-Brown, J.C., Publicover, S.J. Techniques for Imaging Ca 2+ Signaling in Human Sperm. J. Vis. Exp. (40), e1996, doi:10.3791/1996 (2010). Abstract Fluorescence microscopy of cells loaded with fluorescent, Ca 2+ -sensitive dyes is used for measurement of spatial and temporal aspects of Ca 2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca 2+ -reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca 2+ -reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca 2+ response as % change in fluorescence is obtained. Video Link The video component of this article can be found at https://www.jove.com/video/1996/ Protocol Sperm from healthy fertile males, with a normal semen analysis, are normally prepared for imaging as follows. 1. Semen samples are stored at 37°C for no more than 30 min. Cells are isolated from the seminal plasma by swim up into supplemented Earle's balanced salt solution (sEBSS; mM: 1.8 CaCl 2 .2H 2 O, 5.37 KCl, 0.81 MgSO 4 .7H 2 O, 26.2 NaHCO 3 , 1.0 NaH 2 PO 4 .2H 2 O, 116.4 NaCl, 55.6 D-glucose, 2.73 Na pyruvate, 41.8 Na lactate) supplemented with 0.3% charcoal de-lipidated/fatty acid free Fraction V BSA (quality of the BSA is crucial for successful capacitation of sperm). 1 ml of sEBBS is pipetted into each of a series of 5 ml tubes and gently underlayered with 0.3 ml of semen. After incubation for 1 hour (37°C; 6% CO 2 ) the top 0.7 ml is gently removed from each tube and pooled. 10 μl of the sperm suspension is diluted with 90 μl of 1% (v/v) formalin to immobilize the cells, then sperm are counted in a Neubauer chamber. Cell density in the suspension is then adjusted (with sEBSS) to 6 million cells/ml. 2. The sample is then divided into aliquots of 200 μl in loosely-capped tubes and incubated (37°C; 6% CO 2 ) in for 5-6 h to allow capacitation. 3. Coverslips (22x50 mm) have previously been treated with poly-D-lysine. 10 μl of poly-D-lysine solution (10% w/v) is applied as a number of small drops to the centre of the coverslip. The poly-D-lysine is then allowed to air dry. This can be on a heated stage and should be to complete dryness. A coverslip is attached with vacuum grease to an enclosed, purpose-built, perfusable, polycarbonate imaging chamber (dimensions 35 mm x 20 mm x 5 mm; capacity ≈ 180 μl) similar to the Warner RC20 chamber .The poly-D-lysine-coated coverslip forms the base of the chamber when the cells are viewed on an inverted microscope and a 12 mm diameter circular coverslip forms the upper surface of the chamber, allowing transmission of light. 4. Oregon Green BAPTA1-AM (OGB) or Calcium Green 1-AM are used for labeling cells. OGB is prepared by dissolving in DMSO. Pluronic acid F127 (a detergent) is included in the DMSO to prevent 'clumping' of the dye. This can be prepared in the laboratory (100 mg Pluronic in 0.5 ml DMSO), immediately before use, or can be purchased pre-prepared. 20 μl is added to a 50 μg aliquot of OGB (2.5 mg/ml). The vial can then be stored frozen and thawed for later use. 5. After capacitation of the sperm (5-6 h in sEBBS, 37°C; 6% CO 2 ) a tube is selected for imaging and 1.2 μl of OGB solution is added, giving a final concentration of 10 μM. The tube is then incubated for a further 40 min, after which the cell suspension is gently injected into the inflow port of the imaging chamber with a p1000 (blue) pipette tip. The chamber is placed in the incubator, poly-D-lysine-coated coverslip side down, for 20 min. Cells tend to swim along the surfaces of the chamber and will adhere to the poly-D-lysine-coated area. 6. The chamber is now mounted on the microscope, connected to the perfusion apparatus and perfused at approx 0.5 ml/min. A roller pump feeds saline into the chamber and overflow from the exit port is removed by a suction pump with trap. The preparation is left in the dark for at least 10 min whilst loose cells and extracellular dye are removed by perfusion of the chamber. Temperature stability is crucially important