Time-lapse fluorescence microscopy assays were carried out as previously described (Oka et al, 2022 (link)). Briefly, after the cultivation of the X. citri inocula in 2xTY supplemented with the appropriate antibiotics, cells were harvested by centrifugation (5000 rpm, 5 min), washed in water twice and the OD600nm was normalized to 0.5. Then, 1 µL of each X. citri strain were pipetted onto a thin LB-agarose support supplemented with propidium iodide (1 µg/mL), appropriate antibiotic and observed with a Nikon Eclipse Ti microscope equipped with filters for GFP (GFP-3035B-000-ZERO, Semrock) and propidium iodide (TxRed-4040B, Semrock) and a Nikon Plan APO 100x objective. Images were collected every 10 min. Image processing and quantitative analysis of the number of cells having a damaged cell envelope and cell counting related to Movies EV1 –5 were performed manually using Fiji software (Schindelin et al, 2012 (link)) multipoint tool. Time-lapse microscopy was also performed for bacterial competition experiments between X. citri wild-type transformed with pBBR-RFP and X. citri Δ8Δ2609-GFP transformed with pBBR-GFP (Movie EV6 ). Cells were grown and washed as above and the cultures were mixed at a 1:1 ratio and microscopy was performed as described above, in the absence of propidium iodide.
Gfp 3035b 000 zero
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The GFP-3035B-000-ZERO is a lab equipment product manufactured by IDEX Corporation. It is a general-purpose device for performing various laboratory tasks. The core function of this product is to provide a controlled and consistent operating environment for experiments and analyses. No further details or interpretation on the intended use of this product are provided.
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2 protocols using gfp 3035b 000 zero
1
Time-lapse Fluorescence Microscopy of Xanthomonas citri
2
Subcellular Localization of 60S Assembly Factors
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The strains Δnop53/GAL1-ProtA-NOP53 carrying GFP C-terminal fusions chromosomally integrated and expressing the plasmid-borne Nop1-RFP as a nucleolar control were used to survey the subcellular localization of different 60S AFs upon depletion of Nop53. Exponentially growing cells cultured for 18 h in glucose or galactose-containing minimal media were evaluated by fluorescence microscopy. An aliquot of each yeast culture was also analyzed by SDS-PAGE and western blot to confirm Nop53 depletion and evaluate the expression levels of the GFP fusions. Live yeast cells were mounted on agarose pads and imaged using a Nikon Eclipse Ti microscope fitted with a Plan Apo VC 100× Oil DIC N2 objective and an EM-CCD camera (DU-885, Andor) controlled with NIS Elements AR software (version 4.11, Nikon). Images were captured at exposure times ranging from 1 to 3 s, at 13 MHz readout speed, using filters for green (GFP-3035B-000-ZERO, Semrock) and red fluorescence (Texas Red BrightLine set, TXRED4040-B, Semrock) as previously described (69 (link)). Images were processed and analyzed using FIJI (Fiji Is Just ImageJ) 2.0.0-rc-69/1.53c software (National Institutes of Health, Bethesda, MD, USA). More than 150 cells were analyzed in both conditions, in replicate experiments (Supplementary Table S4 ). The RGB profile plots were analyzed using the open-source RGB_Profiler plugin.
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