Cells in the mid-log phase of growth (1−5 × 106 cells/ml) were grown in HL5 overnight to confluency in Petri dishes. The following day, cells were washed twice with KK2 buffer and then submerged in fresh KK2 buffer. Starving cells were imaged using a Nikon Ts2R-FL inverted microscope equipped with a Nikon Digital Sight Qi2 monochrome camera. Cells were also harvested after 4 h of starvation, lysed with NP40 lysis buffer, and analyzed by western blotting to assess the effect of cln5-deficiency on the intracellular and extracellular amounts of CadA, CtnA, and CtsD, as well as the intracellular and extracellular amounts of Cln5 in atg1– and atg9– cells. In separate experiments, 5 × 106 cells were deposited into 6 well dishes and allowed to adhere for 30 min. Cells were then washed twice with FM-aa and incubated in fresh FM-aa for 24 h. Mounds were imaged after 24 h using a Nikon Ts2R-FL inverted microscope equipped with a Nikon Digital Sight Qi2 monochrome camera.
Ts2r fl inverted microscope
Manufactured by Nikon
Sourced in Japan
The Ts2R-FL inverted microscope is a laboratory equipment designed for research and analysis applications. It features an inverted optical configuration and fluorescence illumination capabilities. The core function of this microscope is to provide high-quality imaging and observation of biological samples and other specimens.
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5 protocols using ts2r fl inverted microscope
1
Dictyostelium Starvation and Development
2
Quantitative Cell Imaging in Hydrogels
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Bright field images of cells encapsulated in hydrogels were acquired on a Nikon TS-2R-FL inverted microscope. Quantitative analysis was performed by ImageJ (National Institutes of Health, v. 1.51J8, Bethesda, MD, USA). A paired sample t-test was used to determine the statistical significance.
3
Folic Acid Chemotaxis Assay Protocol
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Chemotaxis toward folic acid was assessed using a radial bioassay (O’Day, 1979 (link)). Briefly, cells in the mid-log phase of growth (1–5 × 106 cells/ml) were grown in HL5 overnight to confluency in Petri dishes. The following day, cells were harvested, washed two times with KK2 buffer, and plated (1 × 108 cells/ml) in 0.6 μl volumes on 0.5% agar/KK2 buffer ± folic acid (50 μM). Cell spots were imaged at 0 and 5 h using a Nikon Ts2R-FL inverted microscope equipped with a Nikon Digital Sight Qi2 monochrome camera. Images were viewed using NIS Elements Basic Research and analyzed using Fiji/ImageJ (Schneider et al., 2012 (link)).
4
Investigating Nedd8 Regulation in Dictyostelium
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Aggregation was assessed as previously described [57 (link)]. Briefly, cells were deposited into 6-well dishes, submerged in HL5, and left to grow overnight. The following day, confluent cells were washed twice with KK2 buffer and then submerged in KK2 buffer ± MLN4924 (1 µM, 10 µM, 50 µM). Cells were imaged using a Nikon Ts2R-FL inverted microscope equipped with a Nikon Digital Sight Qi2 monochrome camera. Images were viewed using NIS Elements Basic Research. To determine the effect of MLN4924 on the amounts of free Nedd8, CmfA, and CadA, cells were deposited into Petri dishes, submerged in HL5, and left to grow overnight. The following day, confluent cells were starved in KK2 buffer ± MLN4924 (50 µM) for 6 h, after which time the conditioned buffer was harvested and cells were lysed with a buffer containing 0.5% NP-40, 150 mM NaCl, 50 mM Tris, pH 7.5, and a protease inhibitor tablet (i.e., NP-40 lysis buffer) (Fisher Scientific Company, Ottawa, Ontario, Canada). Samples were stored at −80 °C for future use.
5
Quantifying SARS-CoV-2 and SARS-CoV Cytotoxicity
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To ascertain cell damage on SARS-CoV-2 and SARS-CoV infection, we quantified cell viability with the CellTiterGlo assay (Promega, Madison, WI, USA). We lysed cells together with culture supernatant at a ratio of 1:1 (volume) with the CellTiter-Glo reagent and incubated this mixture at room temperature for 10 min, then we measured the luminescence signal with the Vector X3 multilabel plate reader (Perkin Elmer). We obtained images of cellular cytopathic effect on SARS-CoV-2 and SARS-CoV infection at 72 hpi with a Nikon Ts2R-FL inverted microscope (Nikon, Tokyo, Japan).
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