To visualize the distribution of radioactivity, the poplars were dried at 60 °C for one day pressed between paper and two glass plates. Autoradiographs were taken with a Phosphorimager (FLA 5100, Fuji, Japan) after exposure for 30 min on imaging plates (Imaging Plate BAS-MS 2040, 20 × 40 cm, Fuji, Japan). The image was taken with the program Image Reader FLA-5000 (version 3.0, Fuji Film, Japan) with 100 μm resolution and analyzed with AIDA Image Analyzer (version 4.27, raytest Isotopenmeßgeräte, Straubenhardt, Germany).
Fla 5000 image reader
Manufactured by Fujifilm
Sourced in Japan
The FLA-5000 image reader is a lab equipment product from Fujifilm. It is designed to capture and digitize images from various types of media, including film, gels, and blots. The FLA-5000 utilizes fluorescence detection technology to generate high-quality digital images for analysis and documentation purposes. The core function of the FLA-5000 is to convert analog image data into a digital format that can be processed and analyzed using specialized software.
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5 protocols using fla 5000 image reader
1
Visualizing Radioactive Tracer Distribution
2
In vitro Munc18-1 Phosphorylation Assay
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HEK293T cells were transfected with Munc18-1. 36 hours after transfection cells were lysed in 1x Laemmli sample buffer (2% SDS, 10% glycerol, 0.26 M B-mercaptoethanol, 60 mM Tris-HCl pH 6.8). After denaturing (boiling for 5 min) the samples were diluted 15x in IP-buffer (50 mM Tris-HCl pH 7.5, 1% Triton-X100, 1.5 mM MgCl2, 5.0 mM EDTA, 100 mM NaCl). Munc18-1 was immune-precipitated with polyclonal Munc18-1 Antibody (cell signalling). ProteinA agarose beads were added and the samples were tumbled for 2 hours at 4 degrees. After 2 hours the precipitations were washed 2x with IP-buffer and 3x with 1x kinase buffer (New England Biolabs). Kinase mixes with either active Dyrk1a (Millipore) or ERK (Sigma Aldrich) were added to the samples (400 ng kinase for each sample). 25 μM [γ-32P]-ATP was added and samples were incubated at 37 °C for 30 minutes. The kinase reaction was stopped by adding 5ul 5x Laemmli sample buffer and samples were boiled for 5 minutes before analysing them with SDS-PAGE. The gels were imaged with the Image Reader FLA-5000 (Fuji).
3
Visualizing Iron Transport in Arabidopsis
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After 24-h Fe starvation, split-root-cultured Arabidopsis plants were subjected to the Fe transport assay. Roots from the left side that were grown in a medium containing sufficient Fe were transferred to a medium with 75 µM Fe labeled with 59Fe (10 kBq). After 24-h incubation, the distribution of 59Fe in the shoot was visualized by radioluminography using an imaging plate (BAS-IP MS 2040, FujiFilm, Tokyo, Japan) and an FLA-5000 image reader (FujiFilm). The amount of 59Fe in each sample was measured using an NaI scintillation counter (ARC-370, Hitachi Aloka Medical, Ltd., Tokyo, Japan) for 10 min. All experiments were conducted with three independent biological replicates.
4
Sodium Uptake and Distribution in Plants
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Two-week-old seedlings grown in the half-strength Kimura B nutrient solution were transferred to the nutrient solution containing 10 mM NaCl for three days. Then, the seedlings were placed in the nutrient solution supplied with 10 mM NaCl and 22Na (18.5 kBq/mL) for labeling. After labeling for 1 h, roots of the seedlings were dipped in the cold nutrient solution for 10 min to remove adsorbed 22Na. The labeled seedlings were cut into roots and shoots, and weighted. The amount of 22Na (cpm) in the root and in the labeled nutrient solution were measured using Gamma Counter (ARC-300, Aloka, Tokyo, Japan), and the amount of Na accumulated during 1 h was determined. The shoots were further separated into each leaf part and the 22Na distribution was quantitatively visualized using an imaging plate (BAS-IP-MS, GE Healthcare, Tokyo, Japan) and a FLA5000 image reader (FujiFilm, Tokyo, Japan). Sodium uptake rate was determined by dividing the total Na amount (nmol) accumulated in the whole plant during 1 h by the weight of the root (mg).
5
Radiolabeling and Imaging of Grafted Plants
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Intact Nb plants, Nb homografts and Nb/At heterografts were conducted to the radio isotope experiments. All leaves except for three to four pieces of leaves in intact or scion plants were removed, and then the stem was cut at its base and placed in a 5 ml tube that contained 2 ml of distilled water, Pi (0.1 µM), and 32 P-phosphate (10 kBq). After 6 h incubation at 27℃, the distribution of 32 P in the plant was visualized by radioluminography using an imaging plate (GE Healthcare UK, Buckinghamshire, UK) and a FLA-5000 image reader (Fujifilm, Tokyo, Japan). The amount of 32 P was calculated with the image analysis software (Image Gauge version 4.0, Fujifilm). For the timecourse analysis, we employed the RRIS (Sugita et al., 2016) , which enable us to observe 32 P distribution in the plant sequentially. The activity of 32 P-phosphate in the incubation solutions was 30 kBq for Nb and Nb/Nb and 60 kBq for Nb/At. The radioactivity image was captured during the dark period of a 15 min light/dark cycle. The accumulation of 32 P in the scion and stock was examined in two sections (10x10 cm in size) collected above and below the graft union, respectively.
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