1. The apoptotic pathway in fertile and subfertile men: a case-control and prospective study to examine the impact of merocyanine 540 bodies on ejaculated spermatozoa 
Nardhy Gomez-Lopez, Ph.D., Guadalupe Estrada-Gutierrez, Ph.D., Alinne Colin, M.D., M.Sc., Arturo Flores-Pliego, M.Sc., Xochitl Flores-Escobar, B.Sc., Sergio Oehninger, M.D., Ph.D., Gerardo Barroso, M.D., M.Sc. 
Fertility and Sterility 
Volume 99, Issue 5, Pages (April 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Determination of early apoptosis/annexin-V binding. (A) Representative plot and histogram showing the parameters used for the flow cytometry analyses. Annexin-V-FITC (FL1 detector)/PI uptake (FL2 detector). The percentage of viable cells is 60.74% [annexin-V(−)/IP(−)]; 34.92% of cells are in early apoptosis [annexin-V(+)/PI(−)]; and 4.34% are necrotic cells [annexin-V(+)/IP(+)]. PI was used to label the DNA in the apoptotic cells. (B) Representative micrographs of stained cells showing viable (empty arrows) early apoptotic (filled arrows) spermatozoa, and (C) necrotic sperm cells. In panel C, the filled arrow shows an M540 body.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Determination of TUNEL and PI or M540 bodies. (A) TUNEL and PI. Representative plot showing the parameters used for the flow cytometry analyses: DNA-PI (FL2 detector) and dUTP-FITC/TUNEL (FL1 detector). The percentage of nucleated viable cells is 83.78% [DNA-PI(+)/dUTP-FITC(−)]; 8.11% are nucleated cells with DNA fragmentation [DNA-PI(+)/dUTP-FITC(+)]; 1.18% are cells devoid of nucleus and with DNA fragmentation (i.e., M540 bodies) [DNA-PI(−)/dUTP-FITC(+)]; and 6.93% are cells devoid of nucleus and without DNA fragmentation (i.e., cell debris) [DNA-PI(−)/dUTP-FITC(−)]. PI was used to label the cellular DNA in viable cells. Representative micrographs show (B) viable sperm and M540 bodies (filled arrows), (C) sperm with DNA fragmentation, and (D) M540 with DNA fragmentation. (E) Determination of M540 bodies and TUNEL. Representative plot showing the parameters used for the flow cytometry analyses: M540 (FL2 detector) and dUTP-FITC (FL1 detector). The percentage of M540 bodies is 1.51% [M540(+)]/dUTP-FITC(+)]; 8.5% are sperm cells with DNA fragmentation [M540(−)/dUTP-FITC(+)]; 0.46% show nonspecific labeling with M540 [M540(+)]/dUTP-FITC(−)]; and 89.53% are viable cells [M540(−)]/dUTP-FITC(−)]. (F) Representative micrographs show M540 bodies (filled arrows), sperm with DNA fragmentation (green arrows), and viable sperm (empty arrows).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions