CH27 cells were maintained in low glucose DMEM (Gibco) containing 15% FBS (Mediatech), 10 mM HEPES, 110 mg/L sodium pyruvate, 50 µM BME, and 1% Pen/Strep in 5% CO2 at 37°C. Cells were plated at 100,000/ml and grown overnight on glass bottom wells (MatTek Corporation) before staining with 5 µg/ml labeled f(Ab)1 for ten minutes and extensive washing with PBS prior to imaging. mEos3.2[16 (link)] tagged Lyn protein was created using standard cloning techniques from Lyn-eYFP[33 (link)] (a gift from Barbara Baird) and mEos3.2 was generated from mEos2[15 ] and was’ a kind gift from Akira Ono. 106 CH27 cells were transfected with 1 µg mEos3.2 tagged Lyn plasmid DNA (a gift from Akira Ono) in Clontech N1 vector (Clontech) using Lonza Nucleofector electroporation (Lonza) for expression of mEos3.2 in Figure 1c .
Glass bottom wells
Manufactured by MatTek
Sourced in United States
Glass bottom wells are a type of laboratory equipment used to hold small volumes of liquid samples. They feature a transparent glass bottom that allows for optical observation and imaging of the samples.
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2 protocols using glass bottom wells
1
Single-Molecule Microscopy of Lyn Protein
2
Live-cell Imaging with Confocal Microscopy
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Cells were grown on glass bottom wells (MatTek, Ashland, MA, USA), and incubated under the conditions described above. During live cell imaging cells were incubated in a PeCon incubation chamber (Erbach, Germany) at 37 °C and 5% CO2. Images were recorded with an LSM 710 confocal laser scanning system (Carl Zeiss Microimaging, Jena) using a Plan-Apochromat 63 ×/1.40 Oil DiC objective.
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