An electronically cooled camera from Princeton Instruments (PIXIS 1024) was used to follow luminescence using the Lightfield program. Camera runs were completed at 25°C, with cultures grown in black, flat-bottomed 96-well plates from Eppendorf. Liquid suspensions of conidia (O.D. of 0.5 at 600nm) were plated onto QA medium (0.03% glucose, 0.05% arginine, 0.001 M QA, pH 5.75) containing 25mM luciferin and covered with a Breathe-Easy strip (USAScientific). Inoculated plates were then subjected to 48 h of 12:12 dark:light cycle conditions at 25°C:28°C before starting the camera trial in constant-dark and 25°C conditions. Signals were accumulated for 15 min every hour. A custom ImageJ Macro called “Toolset Image Analysis Larrondo’s Lab v. 1.0” was used to process the images (Larrondo et al., 2012 (link)) within FIJI v.2.0.0 (Schindelin et al., 2012 (link)). Raw data arising from each time series was smoothed using a 3-point moving average, and then processed using custom-written software to detrend (by removing the exponential decay signal), rescale, and normalize amplitudes.
Flat bottomed 96 well plates
Manufactured by Eppendorf
Sourced in Germany
Flat-bottomed 96-well plates are a common laboratory equipment used for various applications. These plates consist of a grid of 96 individual wells, each with a flat bottom, designed to hold small volumes of liquid samples or reagents. The flat-bottomed design provides a stable surface for various experimental procedures.
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3 protocols using flat bottomed 96 well plates
1
Bioluminescence Assay for Fungal Circadian Rhythms
2
Determining SARS-CoV-2 Infectious Titer
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The infectious activity of SARS-CoV-2 was determined by estimating the CPE on Vero cells in flat-bottomed 96-well plates (Eppendorf, Hamburg, Germany). A series of ten-fold dilutions of the initial virus-containing medium in an SM (1 × 101 TCID50/mL to 1 × 107 TCID50/mL) was prepared. Then, 200 μL of the prepared dilutions was added to each of the seven wells with a monolayer of Vero cells after removing from them the serum-supplemented MEM. At least 12 wells were left for cell culture control. The plates were incubated at 37 °C in an atmosphere of 5% CO2 until the appearance of a characteristic CPE, which was estimated on day 4 using light microscopy. The infectious virus titer (TCID50/mL) was determined using the Reed–Muench method [28 (link)].
3
Induction and Identification of Bregs
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To induce Bregs, DCs (104/well) were cultured in flat-bottomed 96-well plates (Eppendorf, Hamburg, Germany) in complete RPMI overnight at 37°C, and isolated autologous B cells were added to the cultured DCs the next day with or without autologous T cells at a ratio of 1:4:4 (DC:B cell:T cell) in fresh medium with 0.25 µM CpG-ODN (InvivoGen, Toulouse, France). After 7 days of co-culture, proliferation of T cells was assessed by the expression of intranuclear Ki-67. To identify IL-10-producing CD19+ Bregs, co-cultures were incubated for 48 h and thereafter stimulated for 3 h with Cell Activation Cocktail followed by 1 h stimulation with 2 µM monensin prior to intracellular IL-10 staining. The cells were assessed using flow cytometry.
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