FLUIGENT ODROPSEQFPCK Bidirectional Microfluidic Flow Sensor

Imininingwane Yomkhiqizo

• Igama Lomkhiqizo: DropSeq Fluidic
• Inombolo Yengxenye: I-ODROPSEQFPCK
• Inguqulo: Okthoba 20

Ulwazi Lomkhiqizo

The DropSeq Fluidic protocol aims to explain how to replicate the fluidic part of the Macosco DropSeq protocol using Fluigent pressure controllers instead of syringe pumps. The advantage of pressure controllers is their fast response times, enabling the generation of droplets quickly. Pressure control allows for droplets of the right size to be produced in less than two seconds. Additionally, flow sensors enable direct flow control, mimicking the operation of a syringe pump.

Izimpahla nezindlela:
To implement this protocol, you will need the following materials:

• Iphakheji ye-DropSeq
• Umkhiqizo wangaphandle (uyazikhethela)
• Izingxenye ezahlukahlukene njengoba zibalwe encazelweni yokuqukethwe

Imiyalo yokusetshenziswa komkhiqizo

Ukulungiselela Idivayisi Ukuhlolwa Okusha:

1. Hlola amadivayisi we-PDMS ngaphambi kokusetshenziswa.
2. Khipha indawo encane yefilimu emboze amajeneretha.
3. Add a drop of dSURFan  or alternative surfactant into the outlet and wait.
4. Xhuma ishubhu kudivayisi ye-microfluidic.
5. Faka ingcindezi encane usebenzisa i-Flow-EZ.
6. Wait for fluid to come out of the in-/outlets and set pressure back to 0 mbar.
7. Start vortex stirrers and apply pressure to reservoirs until fluid comes out.
8. Xhuma wonke amashubhu ngokuya ngohlelo.

Kwethulwa i-Experiment:
After proper connection, start a DropSeq run by applying pressures to different reservoirs using A-i-O software. Droplets should form in a few seconds.

Ukulawula Ukugeleza:
With a Flow Unit, use the Flow-EZ as a syringe pump. The A-i-O software allowflow ratete commands, adjusting pressures accordingly. Save the pressure configuration once set.

INHLOSO YEPROTOCOL

• This protocol aims to explain how to reproduce the fluidic part of the Macosco DropSeq protocol using Fluigent pressure controllers instead of syringe pumps.
• I-advan enkulutage of pressure controllers is the very fast response times. Using pressure control, droplets of the right size can be generated in less than two seconds. Using flow sensors, it is also possible to use direct flow control in order to use the pressure controller in the same way you would use a syringe pump.

IZIMPAHLA NEZINDLELA

To realize this protocol, you need the following material:

Product                    Reference              Content description
Iphakheji ye-DropSeq	I-ODROPSEQFPCK	3 *Ibha ye-FlowEZ 2
		1 *Isixhumanisi
		1 *Isiqephu esingu-15 ml
		2 *Pcap 2ml
		2 *Iyunithi yokugeleza M
		1 *Iyunithi yokugeleza L
		DropSeq PDMS chip containing 23 designs
		Ikhithi yokuhlinzeka nge-Flow EZ
		2*Low flow rate kit
		Ikhithi yezinga lokugeleza eliphezulu
		DropSeq connecting and tubing kit
Umkhiqizo ozikhethela wona	I-IMCA001	Digital high-speed microscope
	DR-RE-SU-12	12mL dSURF
	I-FLPG+	Umthombo wengcindezi
External product	Barcoded beads	Barcoded beads: Chemgenes CSO-2011*
	I-Vortex mixer	Vortex mixer: Sigma, Fisher Scientific…

The first step is to recreate the following setup.

ISIVIVINYO

Preparing the device for a new experiment
When starting a new experiment, it is recommended to push out all the air from the chip and the tubing. For that, follow the following steps:

1. Before using the PDMS devices, please check the generators carefully. If you have any concerns/questions, please do not hesitate to contact us.
2. Do not fully peel off the film at the beginning: Please cut off the small area for each generator for use. (All of our PDMS chips are covered with Sealing Film, hydrophobic porous sealing film with medical-grade adhesive for tissue culture plates.)
3. Add one drop of dSURF (or Fluorinated oil with other surfactant if not using the dSURF) into the outlet before starting the experiment and wait for a few minutes. Please increase the flow rates of the F-oil and aqueous phases step by step. Avoid jumping from 0 to ~14 mL/h (or maximum flowrates) directly.
4. Connect the dSURF (or Fluorinated oil with other surfactant if not using the dSURF) tubing to the right entry port on the microfluidic device.
5. Apply a small pressure using the Flow-EZ (about 200 mbar should be enough)
6. Wait until you see some oil coming out of the open in-/outlets of the chip and then set the pressure back to 0 mbar.
7. Start the vortex stirrers and apply a small pressure to the two other reservoirs until you see a small drop of fluid coming out of the end of the tube.
8. Stop all pressures and connect all tubings according to the schematic

Iqalisa ukuhlola
Once the device is connected properly, starting a DropSeq run is very fast. Simply apply the following pressures to the different reservoirs using the A-i-O software. The droplets should appear in less than a few seconds.

• P (Oil) = 1300 mbar
• P (Cells) = 540 mbar
• P (Beads) = 580 mbar
  These pressures should generate droplets of about 90-130 µm, which leads to encapsulation rates from 1 to 3 ).
  Ithiphu:
  Using the A-i-O software, pressure configurations can easily be saved. This allows setting all pressures to a given point at the same time.

Ukulawula ukugeleza

• Combined with a Flow Unit, the Flow-EZ can be used exactly as a syringe pump. The A-i-O software allows flowrate commands. A feedback loop on the flowrate then adjusts the pressures to the right value. This technique can be useful to evaluate the necessary pressures for the experiment.
• Once in the right state, the pressure configuration can then be saved.

  

For additional information, contact us by email: contact@fluigent.com or consult our webindawo: www.fluigent.com.

FAQ

How fast can droplets be generated using pressure controllers?

Droplets of the right size can be generated in less than two seconds using pressure controllers.

Can I use direct flow control with this protocol?

Yes, flow sensors enable direct flow control similar to using a syringe pump.

Amadokhumenti / Izinsiza

FLUIGENT ODROPSEQFPCK Bidirectional Microfluidic Flow Sensor [pdf] Incwadi Yeziqondiso ODROPSEQFPCK, IMCA001, DR-RE-SU-12, ODROPSEQFPCK Bidirectional Microfluidic Flow Sensor, ODROPSEQFPCK, Bidirectional Microfluidic Flow Sensor, Microfluidic Flow Sensor, Flow Sensor

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