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Agarose pad mounting protocol for yeast live-cell imaging

For full efficiency of the CherryTemp temperature controller, samples must be placed close to the CherryTemp thermalization chip (<500µm). Therefore, preparing a thin agar pad is a critical step. The following protocol shows how to prepare thin agar pads and how to couple them to the CherryTemp technology

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Figure 1: Align 3 microscope slides and stick lab tape (red in the figure) on the 2 external ones (lengthwise)

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Figure 2: Drop melted agarose (50 to 100µl) on the middle one. Diameter of the pad must be around 1cm diameter

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Figure 3: Top the agarose with another clean slide, placed perpendicularly. Apply gentle pressure and allow agarose to solidify

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Figure 4: After agarose has solidified, gently remove the top slide. Agarose is ready to use and the pad will be less than 500µM width

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Figure 5: Transfer the pad onto the CherryTemp chip (engravements facing down, cover slip and liquid microfluidic chamber facing up). The pad must be centered on the thermalizing pattern

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Figure 6: Pipet the yeast sample on the agar pad. Few microliters of medium is enough. For optimal cell density, first concentrate your sample by spinning cells. Important: yeast cells must be centered on the agar pad

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Figure 7: Top with a high-resolution glass coverslip. The mounted sample is ready for observation in an upright microscope configuration

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Figure 8: Flip the mounted sample 180° for an inverted microscope configuration

Methods adapted from P. Tran lab (Institut Curie) and from our user’s feedback.

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