The microfabrication of the MICS chip was first presented in the supplementary information of Ref. 2. It consists of bonding Metglas foil to a glass wafer, three photolithography steps, dicing the wafer and bonding a PDMS ceiling. A brief review is presented in this protocol.
1. Spin coat epoxy on 100mm soda lime glass wafers.
2. Clamp Metglas 2714A foil to wafer overnight.
3. Take wafers to cleanroom.
4. Rinse wafers with acetone, then IPA, then dry with N2 and bake at 95°C for 5min.
5. Spin coat (300RPM, 20sec) MCC 80/20 primer.
6. Bake for 2min at 95°C.
7. Spin coat (500RPM 10sec, 2000RPM, 30sec) S1811 photoresist.
8. Prebake at 95°C for 3min.
9. Hard contact exposure of first mask, 150 mJ/cm2, i-line.
10. Develop with MF 312 developer for 30sec, rinse with water
11. Etch with Metglas etchant (HCl:H2O2:H2O, 1:4:23) for approximately 8min or until guides are well defined. Pipette bubbles away during etching.
12. Strip remaining resist with AZ300T, rinse in fresh stripper, then acetone, then IPA, N2 dry.
13. Dehydrate wafer at 115°C for 3min.
14. Spin coat Omnicoat (500RPM 5sec, 3000RPM 30sec).
15. Bake at at 115°C for 2min.
16. Spin coat (1000RPM 30sec) SU-8 3010 photoresist.
17. Prebake at 95°C for 10min.
18. Soft contact exposure of second mask, 200 mJ/cm2, i-line.
19. Post-exposure bake, ramp from 65°C to 95°C, hold for 3min then cool gently.
20. Develop in SU-8 developer for 45sec.
21. Hard bake, ramp from 65°C to 150°C, hold for 15min then cool gently.
22. Spin coat (500RPM 10sec, 1000RPM 45sec) SU-8 3050.
23. Prebake at 95°C for 1hr.
24. Soft contact exposure of third mask, 250 mJ/cm2, i-line.
25. Post-exposure bake, ramp from 65°C to 95°C, hold for 6min then cool gently.
26. Develop in SU-8 developer for 6min.
27. Take wafers out of cleanroom.
28. Dice wafers using a dicing saw.
29. Cast PDMS slabs, same size as diced devices, be sure to set on glass slide so surface is smooth. Cure in 70°C oven overnight.
30. Punch holes for inlets and outlets.
31. Plasma treat PDMS in vacuum plasma chamber for 45sec.
32. Place in 10% v/v APTES:DI water solution for 30min.
33. Rinse with DI water and N2 dry.
34. Place treated PDMS onto devices, apply weight and put in in 70°C oven overnight.
1. Lift cells from culture flasks using 0.125% trypsin, wash with PBS and resuspend at a concentration of 107 cells/ml in a solution of HBSS supplemented with 2% BSA.
2. Count cells and measure viability using automated cell counter (e.g. Countess from Thermo Fisher Scientific).
3. OPTIONAL: fix cells using 4% v/v PFA/PBS for 15min at room temperature (RT) or 90% v/v ice-cold methanol/PBS added drop-wise and incubate on ice for 30min.
4. OPTIONAL: permeabilize cell plasma membrane using 0.5% Triton-X-100/PBS for 12min at (RT)
5. OPTIONAL: block cells in 1% v/v FBS/PBS for 30min.
6. Label cells with biotinylated primary antibody as per manufacturer’s instructions.
7. Wash cells 3x in blocking buffer (2% v/v BSA/HBSS).
8. Remove supernatant.
9. Label cells with anti-biotin microbeads. The volumes presented are for up to 10 million cells. If labelling more cells, scale accordingly but if labelling less than ten million cells still use these volumes.
a. Resuspend cells in 80µl of blocking buffer.
b. Add 20µl of anti-biotin microbeads (sufficient for 10 million cells).
c. Incubate at 4ºC for 30min.
10. Resuspend cells in degassed sorting buffer (degassed 2% BSA/HBSS + 3mM EDTA) to desired sorting concentration (1*106 to 5*106 cells/ml).
a. Buffer can be degassed by putting in Erlenmeyer flask under vacuum for 30min.
Chip Set Up
The day before sorting, chips must be degassed by filling with 1% m/v Pluronic® F-108 in DI water and leaving overnight. This process removes any air from the device and lubricates the SU-8 surface. It is suggested that chips be prepared while cells are being labelled to save time.
1. Remove plunger from two 10ml syringes per chip, place in stand and add luer-lock fitting. Label one syringe as “sample” and the other as “sheath”.
2. Connect one inlet tube from chip to “sheath” syringe, allow pressure of degassing solution to backfill “sheath” syringe.
3. Once “sheath” syringe is filled, remove other inlet tube from degassing set-up, add 20 cm of PTFE tubing, connect to the “sample” syringe and allow the height of the buffer in each syringe to equalize.
4. Once solution has equalized, remove most of the degassing solution (leaving a small amount of fluid to prevent air from entering chip).
5. Fill with degassed sorting buffer, allow to flow from inlet syringes through to outlet syringes, flushing out all degassing solution.
6. Meanwhile, for each chip place a 20ml, 10ml.
7. and 3ml syringe in Chemyx syringe pump and add luer-lock fittings. These syringes correspond to low (20ml), medium (10ml) and high (3ml) outlets. A 3D-printed holder can be used to hold up to 5 sets of syringes per pump. Ensure the syringes are fully depressed, the pump has been set to withdraw mode and the 20ml syringe is selected in the pump menu. Select desired flow rate.
8. Connect outlet tubing to outlet syringes, ensure the tube on the outside of the chip is connected to the 3ml syringe, and the inside-most tubing is connected to the 20ml syringe.
9. Run pump for 2min to remove any air in tubing.
10. Place chip on top of magnet, centred overtop of magnetic guides and not on inlets/outlets. Double sided tape can be used to ensure the chip does not easily fall off.
1. Clamp “sample” syringe, remove sorting buffer and add cell solution.
2. Tap syringes to dislodge any bubbles on the sidewalls of the syringes or luer-lock fittings.
3. Ensure that the levels of “sample” and “sheath” fluid in the inlet columns are at the same height (they should remain the same height for the duration of the sort).
4. Complete checklist:
ÿ All tubing is connected to correct size syringes.
ÿ No chips have any bubbles.
ÿ All pumps have been set to “withdraw” mode.
ÿ All pumps have been set to 20ml syringe size.
ÿ All pumps have been set to correct flow rate.
5. Press start on all pumps, sorting will begin.
6. While chips run, continue to check that inlet column heights remain the same, and there are no air bubbles within the chips.
1. Allow chips to run dry (i.e. stop running no sooner and no later that air bubbles start to appear within the chip). Individual chips can be “stopped” by clamping outlet tubing and removing tubing from outlet syringes. This allows the pump to run other chips while disconnecting others.
2. Once all chips are finished, clamp outlet tubes.
3. Remove tubing from all outlet syringes.
4. For each outlet syringe, label a 15ml falcon tube and weigh the tube.
5. Fill each falcon tube with the contents of the appropriate syringe.
6. Weigh the falcon tubes again, the mass difference can be used to calculate the volume of fluid collected, assuming a density of 1g/ml.
7. Take a small (10µl) sample from each tube for automated cell counting and viability measurement.
8. Use remaining cells for future purposes depending on the application of this platform.
1. Calculate the efficiency of each chip (percentage of cells in each outlet) and the recovery (total cells collected/total cells in sample*100%). The efficiency should match previous sorting or flow cytometry experiments, or desired effect should be seen, depending on application (e.g. higher percentage in low outlet for CRISPR KO screen). Cell recovery should be >90%.