Plant material, growth conditions and transgene cloning
Transgenic Arabidopsis plants were used for both sperm cell and pollen isolation. Plants were sown on soil and grown for 8 weeks in short-day conditions (8 h light at 21°C-23°C) and then transferred to long-day conditions (16 h light) to induce flowering. MGH3p::MGH3-eGFP was obtained by cloning 1.2 kb upstream of the MGH3 transcriptional start site, together with the MGH3 genomic sequence without the stop codon, into the pMDC107 vector[25] by gateway cloning. The MGH3 coding sequence contains all endogenous introns, which might help for stable transcription and accumulation to high levels. Primers used for MGH3p::MGH3-eGFP cloning are presented in Additional file1. Double homozygous plants harbouring MGH3p::MGH3-eGFP and ACT11p::H2B-mRFP[17] transgenes, were obtained by crossing individual homozygous lines. This seed stock is available from ABRC under stock number CS67829.
Purification of sperm cells, vegetative nuclei and sperm nuclei
Open flowers were collected into a 50 ml falcon tube, filled roughly with a volume of 10 ml of fresh material. Flowers were vortexed at medium speed (Fisher Scientific, TOP-mix 3 IKA, speed 2000) in 10 ml of Galbraith buffer (45 mM MgCl2, 30 mM Sodium Citrate, 20 mM MOPS, 1% Triton-100, pH to 7.0) for 3 minutes, at room temperature. This crude fraction was then filtered through a Miracloth mesh (Calbiochem) to remove flower parts and centrifuged for 1 minute at 2600 g to pellet pollen. The supernatant was carefully removed with minimal disturbance and the pollen enriched pellet was resuspended in approximately 1.5 ml of fresh buffer. This pollen enriched fraction was then transferred to a 1.5 ml eppendorf tube containing 100 μl of acid-washed glass beads (425–600 μm, Sigma), and vortexed continuously at maximum speed (2500) for 4 minutes in order to break mature pollen grains. The fraction containing the released nuclei was then filtered through a 28μm mesh (SEFAR) to exclude unbroken and hydrated pollen. The nuclei enriched solution was ready for FACS at this point, which included debris from broken pollen. Hydrated pollen that remained intact and thus was retained in the filter can be recovered by washing the 28 μm mesh in new buffer and used in a second extraction step with the glass beads.
If intact and viable SC are desired for downstream applications (e.g. RNA extraction or in vitro manipulation) the whole procedure should be performed with sperm extraction buffer (SEB) (1.3 mM H3BO3, 3.6 mM CaCl2, 0.74 mM KH2PO4, 438 mM sucrose, 5.83 mM MgSO4, 7 mM MOPS at pH 6), which assures that 75-80% of the sorted sperm cells are viable and intact.
Purification of Microspores from young flower buds
Closed flower buds (approximately 20 ml) were gently ground using mortar and pestle in 10 ml of pollen extraction buffer (PEB) (10 mM CaCl2, 2 mM MES, 1 mM KCl, 1% H3BO3, 10% Sucrose, pH 7.5) in order to release the spores. This crude fraction was initially filtered through Miracloth (Calbiochem) to remove bigger debris, and concentrated by centrifugation (800 g, 5 min) in 15 ml falcon tubes. The resulting yellowish pellet enriched in microspores was resuspended in 1.5 ml of PEB, and filtered through a 20 μm mesh (Partec, CellTrics) before FACS to further enrich for microspores and exclude tricellular and mature pollen.
Fluorescence activated cell sorting
Fluorescent activated cell sorting was carried out with a MoFlo (Beckman Coulter, Fort Collins, USA) with a 488 nm laser (200 mW air-cooled Sapphire, Coherent) at 140 mW used for scatter measurements (Low angle or Forward Scatter, and High angle or Side Scatter; FSC and SSC, respectively) and for GFP excitation, and a 561 nm laser (50 mW DPSS, CrystaLaser) at 38 mW for RFP excitation. GFP and RFP were detected using a 530/40 nm and a 630/75 nm bandpass filters, respectively. FSC was used for triggering, and threshold had to be low to avoid missing the sperm cell population, whose size is on average 2.5 μm in diameter. Phosphate Buffer Saline (PBS) was used as sheath, and run at a constant pressure of 400 kPa (~60 psi). Frequency of drop formation was approximately 96,000 Hz. Even though pollen was present in the sample (broken and intact) it did not interfere with drop formation or break-off, as we were able to sort pollen under the same conditions as for SC and VN. Sorting rates were typically 2 million SC and 1 million VN per hour, i.e. an average rate of 500 SC and 250 VN per second, respectively.
Viability tests were performed with approximately 50.000 sorted SC and VN by staining with SYTOX Orange (Molecular Probes, Invitrogen) at a final concentration of 25nM. Sperm cells negative for SYTOX Orange and positive for GFP were considered viable (intact membrane) and considered compromised or to represent bare nuclei (only the nuclei stained), if positive for both SYTOX and GFP. SYTOX Orange was excited with the 488 nm laser and detected with a 580/20 nm bandpass filter in the Moflo.
Purity was determined by running aliquots of sorted cells in a CyAn ADP flow cytometer (Beckman Coulter, Fort Collins, USA). A 488 nm laser was used to excite both GFP and RFP, detected with 530/30 and 616/21 nm bandpass filters, respectively. 1 μM DAPI (Sigma) was added to the sorted cells and incubated on ice for 5 min, to discriminate between nuclei containing cells and electronic noise. DAPI was excited with a 405 nm laser and detected with a 450/40 nm bandpass filter.
Microspores were also purified by FACS using a Moflo high-speed cell sorter. The machine was used in a standard configuration, using a 100 μm ceramic nozzle with PBS running at a constant pressure of 200 kPa (~30 psi), and a drop-drive frequency of approximately 30,000 Hz. The 488 nm laser line was used for scatter measurements and autofluorescence excitation, which was detected in the GFP channel using a 530/40 nm bandpass filter. Microspores and other stages of pollen development were identified by their elevated high angle scatter (SSC) and autofluorescence properties (observed in the GFP channel). Within this population, microspores were selected by their characteristic smaller size, captured by a diminished low angle scatter (FSC) and time-of-flight (Pulse Width).
RT-PCR
Semi-quantitative RT-PCR was performed with total RNA isolated from approximately 100,000 cells/nuclei that were sorted directly into Tri Reagent LS (Sigma). First-strand cDNA (Oligo-dT primed) was synthesized in 25 μl reactions using the MLV reverse transcriptase - RNaseH minus (Promega) according to manufacturer instructions. 2 μl of non-diluted cDNA was used as a template for 30 PCR cycles. Primers used are listed in Additional file1.
Comments
View archived comments (2)