Difference between revisions of "Microfluidics"
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** Slowly flow PBS through chip | ** Slowly flow PBS through chip | ||
== Cell filling == | == Cell filling == | ||
+ | * Follow protocol for [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Cell_lab_431#Cell_culturing cell passaging]] | ||
+ | * Transfer the some of cell suspension to a sterile centrifuge tube of appropriate size (Eppendorf or other depending on volume) | ||
+ | * Centrifuge for 10 minutes at 800 × g. | ||
+ | * Note: Certain cell lines are very sensitive to centrifugal force. | ||
+ | * Carefully remove the supernatant without disturbing the cell pellet. | ||
+ | * Add the desired volume of fresh medium gently to the side of the tube and slowly pipette up and down 2 to 3 times to resuspend the cell pellet. | ||
+ | * Transfer the cells to the sterilized microfluid chip | ||
= Running experiment = | = Running experiment = |
Revision as of 13:30, 6 April 2022
Contents
Designing Microfluidic masks
- Stanford microfluidics foundry has a good guide for designing your own device. That includes a guide to use AutoCAD.
- AutoCAD (a program from Autodesk) is available free.
- You can use Klayout but we have more support for using Autocad.
- Mask template for placing your designs
- This guide for designing masks has some important tips
- All fluid pathways have to be inside one or more closed contour(s)
- The outer contour should be drawn in one layer (Give it a name like "Flow")
- Any obstacles inside this outer contour must be drawn in another layer. Give it a name like "Flow inner polygons")
- Displaying your design as PDF or otherwise is not straight forward because the resolution needed. You get a fair impression using CloudConvert which is much better than AutoCads own pdf export.
- Example of a design where white lines (in screen shot below, black lines in PDF) are contours containing flow and green lines are inner polygons. Here is a PDF version of the file and the DWG file.
- Rounding corners on a region
- Command EXPLODE makes region into lines
- Command JOIN joins lines into polylines
- Command FILLET, downarrow to get options RADIUS and POLYLINE
Photolithography
PDMS casting
- PDMS preparation
- Bring cup with PDMS close to wafer in Petri dish
- Pour slowly and avoid introducing air bubbles
- Remove air bubbles by puncturing them
- Fill petri dish to 5-10 mm above wafer surface
Assembling the chip
- Clean glass slide with IP and N2
- Punch holes for fluid inlets
- For 1/16" tubes use OD?? punch
- For thin teflon tube use OD?? punch
- Place both PDMS and glass slide with clean sides up on glass plate in plasma cleaner
- Follow instructions for Plasma treatment
- Place the PDMS on top of the glass slide and watch it attach
- If necessary, apply a gentle pressure to the PDMS
Filling the chip
For sterilizing and filling the chip with water and fibronectin you need
- 1ml syringe with
- 20G needle and
- 2cm long 1/16" teflon tube on the needle.
- small beaker
- ethanol
- distilled water
- Eppendorf tube of fibronectin
- Cells in fresh media
- Inlet and outlet tubes
- Measure lengths of inlet and outlet tubes and cut
- Sterilize the tubes
Sterilizing
- If you want a sterile chip move each object into LAF bench once they have been sterilized
- Spray chip with ethanol and rub off glass side
- Assemble syringe, needle and teflon tube
- Spray ethanol and rub outside
- Rinse beaker in ethanol and pour in a small amount of ethanol
- Retract syringe half way in air, and fill teflon tube with ethanol
- Empty syringe of ethanol and air, make sure it is dry
- Empty beaker of ethanol
Water filling
- Fill a small amount of distilled water in beaker
- Retract <1ml distilled water into tube/needle/syringe
- Fit in inlet of chip and fill slowly
- Empty syringe and needle to be ready to use for fibronectin
Fibronectin filling
- Take one Eppendorf with 0.5 ml fibronectin form freezer
- Thaw the fibronectin
- Retract fibronectin into tube/needle/syringe
- fit in inlet of chip and fill slowly
- Attach inlet and outlet tubes
- Incubate for 30 min in incubator
- Rinse with PBS
- Details?
- Pour ~?? ml PBS in sterile beaker
- Fill sterile ?? ml syringe+needle with PBS
- Slowly flow PBS through chip
Cell filling
- Follow protocol for [cell passaging]
- Transfer the some of cell suspension to a sterile centrifuge tube of appropriate size (Eppendorf or other depending on volume)
- Centrifuge for 10 minutes at 800 × g.
- Note: Certain cell lines are very sensitive to centrifugal force.
- Carefully remove the supernatant without disturbing the cell pellet.
- Add the desired volume of fresh medium gently to the side of the tube and slowly pipette up and down 2 to 3 times to resuspend the cell pellet.
- Transfer the cells to the sterilized microfluid chip