- HOG and ARO bandwidth experiments
Microfluidic chips are made by curing PDMS, following is a the protocol used for bandwidth experiments (Y-Shaped channels).
- Channel height : 42 μm
- Channel length : 1000 μm
- PDMS ratio: 10 to 1
- Mix PDMS and the curing agent at a ratio of 10:1 (54 grams to 6 grams for large petri dishes)
- Vacuum seal to remove bubbles (~30 minutes or more)
- Pour onto wafer, move/remove remaining bubbles with a pipette tip
- Cure at ~60°C for > 2 hours
- Cells streaked on YPD plates 3 days prior to planned experiment
- Cells inoculated in overnight culture in LFM + Urea ~48 hours before experiment at room temperature
- 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
- 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
Following is the way I have been setting it up, it is by no means the only way to do it.
- 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
- Flush flow cell with ~ 0.5 ml Ethanol followed b ~ 0.5ml water in order to clean it
- Fill with ~ 0.5 ml unthawed ConA and allow to incubate for at least 5 minutes
- Flow in ~ 0.5 ml concentrated cells and allow to settle/stick for at least 10 minutes
- 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.
- Allow the flow to reach the end of the outlet tube before starting experiments
- Choose ~5 xy-position/frames to ensure enough cells (there should be ~20-30 cells per frame)
- Check to make sure that the mCherry signal changes by testing the switch manually
- 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
- Place some notes here for visitors
- Example: This project is currently on hold until further notice.