Hi, I would suggest that your sperm fluorescence is low because of poor uptake of PI but that does not mean that you can't make it work. First thing is to increase your PI concentration we have used up to 50 micrograms per ml for mammalian sperm with reasonable results.. However, if your preparation is messy you may have trouble isolating the signal from noise. Not being familiar with ant reproductive anatomy I have no idea how you are collecting sperm (I'm assuming that you aren't just giving them a very tiny specimen jar and some provocative ant literature...). If you are collecting samples from dispersed tissue you may want to try gating or other means to enhance your signal. For mammalian sperm analyses we gate tightly on forward vs side scatter signature before any analyses to get rid of junk. Also, can I suggest that, instead of isolating nuclei, try sonicating your sperm sample using a microprobe sonicator. We do this before flow analyses of rat sperm and it works well in removing the tails AND all other cells in the preparation. Mammalian sperm nuclei are highly compacted and resist this treatment. You may want to test this method prior to any large scale work. In addition, you can try using and alternative stain/procedure to use the highly condensed nature of your targets to isolate them. Use Evenson's SCSA method (e.g. Evenson DP. Flow cytometric analysis of male germ cell quality. Methods Cell Biol 33:401-410 (1990).; Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, De Angelis P, Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 14:1039-1049 (1999).) which uses stains acid treated sperm using acridine orange. AO is a nucleic acid specific stain that fluoresces green when bound to doubles stranded and red bound to single stranded nucleic acid. The ratio of green vs red fluorescence will help you gate junk from cells (only sperm will stain highly green). Good luck! Michael Wade, PhD Research Scientist Systemic Toxicology & Pharmacokinetics Section Environmental Health Program Health Canada Rm. 200 Environmental Health Centre P.L. 0803D, Tunney's Pasture Ottawa, Ontario, K1A 0L2 Canada Ph: 613 946 5127 Fax 613 957 8800 Mike_Wade@hc-sc.gc.ca Michiel Dijkstra <MDijkstra@bi.ku. To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> dk> cc: Subject: Request help for PI-staining of ant sperm 2005-10-31 06:47 AM Dear all, I am new to the field of flow cytometry. We are trying to do ploidy analysis of sperm of diploid vs. haploid male ants. We currently use Vindelov's method and measure the nuclei on a FACSCalibur that is not set up for DAPI, so we use PI. The problem is that the sperm have insufficient fluorescence for the cytometer (even though they light up brightly red under a microscope!!!), presumably due to the high chromatin density in the sperm which interferes with dye uptake. Also, all the "nuclei" remain rod-shaped (as opposed to round) and about 50% have kept their tail. It seems our Vindelov protocol (Basic protocol 2 in section 7.5 of Current Protocols in Cytometry, 1997) is not rigorous enough. We found the protocol below for de-condensing sperm chromatin in Larsen et al. (2004) Theriogenology 62: 501-511. But that protocol is for DAPI staining. So my two questions are: 1. Could we copy the protocol below, just replacing DAPI with PI? Or does the cell lysis procedure depend on the dye that one uses? 2. How is it possible that PI stained "nuclei" are bright red under the microscope, but don't fluoresce enough to be detected on the cytometer? Any other tips would be greatly appreciated. Best wishes, Michiel Dijkstra "The staining procedure in brief: 1 ml fixed sperm cells were transferred to a 10 ml conical test tube and washed once by adding 9 ml PBS and centrifuged (10 min, 200¡Ág, 4 ¡ãC). The supernatant was carefully removed leaving a residue of about 100 ¦Ìl. The cell pellet was dissolved and 5 ml DTE/papain solution (1,4-dithioerythrit 40 mM (Boehringer Mannheim 223,662), papain (Roth 8933.1) 1%, DMSO 2%, in 90 mM Walpoles acetate buffer at pH 5.5) were added and incubated at 20 ¡ãC with slow stirring. After exactly 20 min incubation samples were centrifuged (10 min, 200¡Ág, 4 ¡ãC) in a precooled centrifuge, the supernatant was carefully removed leaving a residue of about 0.1 ml. The nuclei were brought into suspension and 5 ml tris-buffered DAPI (DAPI 1.75 ¦Ìg/ml, NaCl 0.1 M, trisodium citrate 40 mM, in Tris¨CHCl buffer 0.1 M at pH 7.4) was added. The cells were incubated at room temperature for 1 h protected from light before onset of measurement."Received on Tue Nov 1 16:18:00 2005
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