P-CAGE-26805 - P-CAGE-26805
Isolation of Arabidopsis Sperm Cells To obtain sperm cells from mature pollen, freshly opened flowers from the transgenic line AtGEX2::eGFP were collected and placed in a humid chamber at room temperature for 1 hour to hydrate. The sperm extraction buffer (1.3 mM H3BO3, 3.6 mM CaCl2, 0.74 mM KH2PO4, 438 mM sucrose, 7 mM MOPS, 0.83 mM MgSO4, in double distilled water), was adjusted to pH 6.0 with NaOH, filter-sterilized (0.22 µm filtration, 150 mL Bottle Top Filter with a 45 mm neck (Corning Incorporated, New York, USA) and vacuum-degassed. Inflorescences were immersed and mixed in 500 mL of sperm extraction buffer, until the pollen grains were released, yielding a yellowish solution. To remove flowers and other plant tissues, the solution was filtered through Miracloth (Calbiochem®, USA), then the pollen was concentrated by passing the filtrate through a 10µm pore size mesh filter (SEFAR AG, Heiden, Switzerland) using a vacuum pump (KNF Aero Mat, USA). This material collected on the filter was subsequently washed with 1-2mL of buffer, to release pollen and impurities, and then filtered through a 28µm mesh filter (SEFAR AG, Heiden, Switzerland) to dispose of debris larger than pollen. Pollen grains were disrupted using a 2ml glass homogenizer (Kontes Glass Company, Tissue Grind Pestle SC and Tissue Grind Tube S2, Vineland, New Jersey, USA), by 3 circular movements up and down, using 2 mL of sperm extraction buffer at a time. The resulting solution contained free sperm cells as well as burst and intact pollen grains, and was filtered through a 15µm pore size mesh filter (SEFAR AG, Heiden, Switzerland) in order to eliminate most of the debris and the remaining intact pollen grains. Sperm cells were incubated with 1µM DRAQ5TM (Biostatus Limited, Alexis Corporation, Lausen, Switzerland) in order to stain DNA, and the sample was stored at 4ºC before fluorescence-activated cell sorting. Fluorescence Activated Cell Sorting Fluorescence-activated sperm cell sorting was performed in a Moflo High-Speed Cell Sorter (Dako-Cytomation Inc. Fort Collins, CO, USA), using a 100 m ceramic nozzle with 30 psi sheath pressure, a 488 nm laser line from a Coherent Sapphire 488-200 CDRH laser for eGFP excitation, and a 632.8 nm laser line from a Spectra-Physics 107B 25mW HeNe laser to excite DRAQ5. GFP and DRAQ5 were detected using a 530/40 nm and a 700/75 nm HQ band pass filter, respectively. Sperm extraction buffer was used as the sheath solution, both for hydrodynamic stability and subsequent analysis of sorted sperm cells. For subsequent RNA extraction, sperm cells were sorted directly into RNeasy extraction buffer RLT (Qiagen, Germany).