CLINICS 2011;66(7):1237-1245 The therapeutic effect of PEMF on reproduction Kumar S et al. Figure 1 - Assay design for our experimental work. minimum detectable dose of rat melatonin is typically less than 1.02 pg/mL. Creatine Kinase Assay A CK kit was used. The seminal plasma was removed by washing the sperm sample with ice-cold imidazole buffer (0.15 M NaCl and 0.03 M imidazole (pH 7.0) at a ratio of 1515). The supernatant was decanted after centrifugation at 500 g, and the pellet was re-suspended in a 0.1% Triton X- 100 detergent solution by vortexing for 20 seconds. The sample was centrifuged again at 500 g, and the CK activity of the supernatant was analyzed. In this assay, the creatine is enzymatically converted into sarcosine, which is then specifically oxidized to generate a product that converts a colorless probe to an intensely red product. This final product is detected colorimetrically (lmax = 570 nm). The CK activity is expressed in international units/108 spermatozoa. Measurement of Caspase 3 Activity The activity of caspase 3 was also measured using an assay kit. The spermatozoa were centrifuged at 300 g for 10 min at 4˚C. The pellet was then re-suspended in lysis buffer for 20 min and centrifuged at 20,000 g for 20 min at 4˚C, and the supernatant was collected. The assays were conducted in 96-well plates. The colorimetric caspase 3 assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), which results in the release of the p-nitroaniline (pNA) moiety. To assess the specific contribution of caspase 3 activity, the Ac-DEVD-pNA substrate (2 mM) was added to each well according to the instructions of the manufacturer. 1239 The plates were incubated overnight at 37˚C to measure the caspase 3 activity. The absorbance was measured using a microplate reader (Spectromax M2) at 405 nm. The caspase 3 activity is expressed in mmol of released pNA per min per ml of cell lysate at 37˚C. Testosterone Assay A serum testosterone assay was performed using the testosterone EIA kit (Cayman, USA). The procedures for the assay were followed as described in the manual of the manufacturer. In brief, 50 ml of the testosterone standard or serum, 50 ml of the testosterone AChE tracer, and 50 ml of the testosterone antiserum were added to the wells of an ELISA plate containing 100 ml of EIA buffer. The sensitivity of the assay was 6 pg/ml. The optical density was read using a spectrophotometer (SpectroMax M2) that was sensitive at a wavelength range of 405-420 nm. Pulsed electromagnetic field therapy Immediately after exposure to the 2.45-GHz radiation, the Group-V animals (MW + PEMF) were exposed to PEMF for two hours daily. Three animals were exposed at a time. The rats were placed into a cage (28661726115 mm) ventilated by one-cm-diameter holes. The animals were exposed in a mu-metal box at 100 Hz (Pulsed Magnetic Field Inventors Groups for Lawson Health Research, London, Ontario). The Mu box (38663336206 mm) was lined with opaque black plastic and fitted with acrylic rods to transmit light into the enclosure (Figure 3). The box was made of 1.6-mm-thick mu-metal (Magnetic Shield, Bensenville, IL), with four rectangular Merritt-like configurations in the box. The coils