User:Bohao Liu/Notebook/Research

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Bandwidth Experiments

  • HOG and ARO bandwidth experiments

Microfluidics

Microfluidic chips are made by curing PDMS, following is a the protocol used for bandwidth experiments (Y-Shaped channels).

Chip specifications

  • Channel height : 42 μm
  • Channel length : 1000 μm
  • PDMS ratio: 10 to 1

Protocol

  1. Mix PDMS and the curing agent at a ratio of 10:1 (54 grams to 6 grams for large petri dishes)
  2. Vacuum seal to remove bubbles (~30 minutes or more)
  3. Pour onto wafer, move/remove remaining bubbles with a pipette tip
  4. Cure at ~60°C for > 2 hours

Cell Culturing

ARO experiments

  1. Cells streaked on YPD plates 3 days prior to planned experiment
  2. Cells inoculated in overnight culture in LFM + Urea ~48 hours before experiment at room temperature
  3. Cells set back 16 hours before experiment at 1:100 in order to achieve an OD of between 0.1 and 0.3 at the time of experiment
  4. In order to get a higher cell density in the actual flow cell, spin cells down (3 ml) at 8000 rpm for 2 minutes and then re-suspend in 500 μl of LFM + Urea prior to loading into the flow cell

Experimental Setup=

ARO experiments

Following is the way I have been setting it up, it is by no means the only way to do it.

Switch
  • LFM + Urea in the switch with the higher pressure on Off and the lower pressure on On
  • LFM + Urea + Tyrosine with .0005 mg/ml rhodamine tracer
Flow Cell
  1. Flush flow cell with ~ 0.5 ml Ethanol followed b ~ 0.5ml water in order to clean it
  2. Fill with ~ 0.5 ml unthawed ConA and allow to incubate for at least 5 minutes
  3. Flow in ~ 0.5 ml concentrated cells and allow to settle/stick for at least 10 minutes
  4. Plug in LFM + Urea port first and make sure that the switch is in the Off position before plugging in the LFM + Urea + Tyrosine port in order to ensure that the cells are not being activated before they are supposed to be.
  5. Allow the flow to reach the end of the outlet tube before starting experiments
  6. Choose ~5 xy-position/frames to ensure enough cells (there should be ~20-30 cells per frame)
  7. Check to make sure that the mCherry signal changes by testing the switch manually

Microscopy Parameters

Aro Experiment
  • mCherry at 200 mS exposure and ND of 2
  • GFP at 200 mS exposure and ND of 2
  • Image taken every 10 minutes for a total of 3 hours, mCherry first then GFP

Notes

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  • Example: This project is currently on hold until further notice.


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