M04G Microfluidic Gradient Plate
M04G-02
Description: The M04G plate contains 4 independent chambers, each with 4 switchable upstream solution channels. The chamber design allows culture of cells (2D or 3D) with exposure to stable diffusion gradients. Each chamber allows for 4 different spatial gradients and is designed for long term, high magnification, live cell perfusion imaging. Applications:  High magnification imaging of cells in a stable diffusion gradient (cell migration, chemotaxis, polarization)  Long term continuous perfusion experiments (3+ days)  Gradient switching experiments (induction, inhibition, drug dosing, etc.)  Comparing up to 4 different cell types or exposure conditions in parallel  Temperature and gas atmosphere control (temperature shift, anoxic, etc.) Product Specifications: 
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Use with ONIX‐262 system and F84 manifold SBS standard footprint frame (fits to typical 96 well stage holders) #1.5 thickness (170 um) glass slide bottom Microfluidic perfusion barriers enable long term spatial gradients Laminar flow rates of 1‐50 ul/hr Microfluidic gas exchange channels Plate Design: A Flow Inlets Outlets Air Channels Culture Chambers (A‐D) B C D 1 2 3 4 5 6 7 8 The M04G has 4 independent units (A‐D), each with 4 inlet wells (2‐5), cell inlet (6), cell outlet (8), cell wash (1), and flow outlet well (7). All four culture chambers are located under a single imaging window. The 4 culture chambers are located under a large imaging window for high magnification phase objectives and to minimize travel distance. All channels are resistance matched for uniformity. Flow Inlets Perfusion Barriers 2 V1 V2 V3 V4 V5 V6 V7 V8 3 1 Lines to Controller Outlet 10 Cell Inlet Gas Inlet 50 um height Cell Out Outlet 4 5 The F84 manifold has 8 pressure channels (V1‐V8) to control flow rates through the microfluidics. A vacuum line is used to seal the plate to the manifold, and a gas line enables atmosphere control. The culture chamber is 4.0 x 0.5 mm in area with a height of 50 microns. Perfusion barriers bound the chamber on the top and bottom edges to separate the chamber from flow channels. 2544 Barrington Court | Hayward, CA 94545 | www.cellasic.com | 510-785-9846
Spatial Gradient 
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Diffusion across perfusion barriers create a stable spatial gradient in the culture area. Continuous flow “infinite source/sink” design maintains stable gradient profile for days. Flexible format allows gradient switching, turning on/
off gradients, and toggling between gradient and single solution exposure. Measurement Line 
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The M04G plate is designed to maintain stable diffusion gradients in 2D or 3D culture during live cell imaging. 0.5
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Stable diffusion gradient at 1 psi. Scalebar = 100 um Solution Switching Our advanced perfusion control enables experiments not possible with existing instrumentation. 
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Four switchable inlet wells for each chamber. Perfusion barrier prevents cells/gel from blocking flow. Short distance between flow channels and culture chamber allows rapid diffusive transport to cells. Perfusion rates of µl/hr allow continuous multi‐day experiments on your microscope. Reversal of a diffusion gradient within 20 minutes. The M04G allows active switching between up to 4 gradients. Highest Quality Live Cell Imaging CellASIC’s microfluidic cell culture technology delivers unmatched quality for live cell imaging. 
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The 50 µm tall chamber with #1.5 thickness (170 µm) glass coverslide floor enables imaging with high NA objectives. Stress‐free cell loading using a pipette inside your sterile hood. Gravity driven perfusion lets you “pre‐culture” cells for many days in a standard incubator. A large imaging window allows light to pass freely for optimal phase contrast quality. The four microchambers are located within a 1.2 cm area, minimizing stage travel and focus drift. HeLa cells immunostained within the microchamber. Long Term Cell Culture The innovative microfluidic plate is designed for long term cell happiness. 
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Continuous perfusion ensures fresh medium and waste removal, even at high cell densities. Advanced chamber design ensures low stress culture environment. The sealed manifold delivers 5% CO2 through specialized microfluidic gas diffusion channels. Gravity driven medium channel allows pump‐free culture in any incubator. HL‐60 neutrophils migrating in response to a gradient. Courtesy Lim Lab, UCSF. 2544 Barrington Court | Hayward, CA 94545 | www.cellasic.com | 510-785-9846
Flow/Cell Inlets Flow Outlet 1 2 3 4 5 6 7 Cell Outlet Operation Instructions 1. The M04G microfluidic plate contains 4 independent chambers, each with 4 solution inlets (Figure 1). Each row of wells (A‐D) addresses the corresponding chamber. The plate is shipped pre‐
primed with sterile PBS. 8 A B Chamber Coating (Optional) C 2. The culture chamber can be coated with standard treatments by adding coating solution to well 6 (cell inlet) and flowing at 1 psi for the desired time period. D Cell Loading 2 3 7 1 8 6 1 7 4 5 1 = Cell Wash 2‐5 = Flow Inlets 8 = Cell Out 6 = Cell In 7 = Outlet 3. Fill inlet wells with 300 µl of desired culture media. Wells 2 and 3 will flow to the top channel and 4 and 5 flow to the bottom channel of each chamber. Flow of one solution from the top and another from the bottom creates a gradient (Figure 3). Up to 4 gradient combinations are possible in the M04G (2+4, 2+5, 3+4, 3+5). 4. Prepare a cell suspension of 1‐4x106 cells/ml. 5. Aspirate the cell loading well (6), cell outlet (8), cell wash (1), and outlet (7) wells. Note: there is a hydrophobic ring at the bot‐
tom of wells 1, 6, and 8. Remove the liquid in the hole at the bottom of wells 6 and 8. Make sure the hole of well 1 is filled with PBS, or replace with your preferred loading buffer. Figure 1. Plate Layout 6. Pipette 5 µl of cell suspension into the hole at the bottom of the cell inlet well (6). Figure 2. Cell Loading Profile Continuous Flow Through Channels Diffusion in to Chamber Figure 3. Stable Gradient Creation 7. Seal the microfluidic plate to the F84 manifold: Clean the mani‐
fold gasket with 70% ethanol and blot dry. Place the microfluidic plate on a flat surface. Align and set the manifold over the wells of the plate. Turn on the vacuum switch on the ONIX box and push down on the manifold with slight force for ~5 seconds to ensure uniform contact during sealing. When a proper seal is formed, the green “sealed” light will be lit. Make sure a proper seal is formed before proceeding. Leave the vacuum on during the course of the experiment. (If the seal is unsuccessful, turn off the vacuum switch and wait for the blue “ready” light to turn back on. Repeat the protocol above. If a seal cannot be formed, please contact CellASIC.) 8. Open the ONIX FG software on a computer attached to the USB line from the control box. Select the “M04” tab from the front page. If you do not see the tab, make sure you have an updated version of the software (www.cellasic.com/Products‐downloads) or contact CellASIC. 9. Use the “Cell Load” tab to introduce cells into the chamber (flow at 0.5 PSI of channels 1 and 6 for 0.5 minutes). Cells should fill the center of the chamber and exit towards well 8 (Figure 2). 10. Optional Pre‐Culture. Unseal the manifold and add 300 µl of cul‐
ture medium to well 1 and 30 µl in well 8 to initiate gravity driven perfusion. Place the plate in a standard incubator. Re‐
place the medium in well 1 every 3‐4 days for long term culture. 2544 Barrington Court | Hayward, CA 94545 | www.cellasic.com | 510-785-9846
Live Cell Imaging 11. Place the sealed plate/manifold assembly on an inverted micro‐
scope (Figure 4). Focus on the center of the imaging area. The culture region is 4.0 x 0.5 mm in size with a 50 micron ceiling height. 12. Fill the four sets of flow inlet wells (col 2‐5) with 300 µl of solu‐
tion. Empty well 7. Make sure wells 1, 6, and 8 are empty (except for the hole within the hydrophobic ring). If less than 4 units will be used, fill the unused inlet wells with buffer to pre‐
vent dehydration. Figure 4. Plate and Manifold on Microscope Stage 13. Flow properties are given in Figures 5 and 6. Figure 5 shows the total flow rate out of each inlet well (2‐5). Solutions will diffuse through the perfusion barriers at the top and bottom of the chamber. Figure 6 gives the diffusion time to reach the center of the chamber for solutes of varying diffusivities. (Most proteins have diffusivities ~1E‐6 cm2/s, and glucose is ~5E‐6 cm2/s.) 14. Use the tabs on the right side of the software control window (Figure 7) to set up your experiment. Flow Rate (ul/hr)
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15. For custom flow scheduling, use the “Protocol” tab to enter text based commands. The commands are given in Figure 7. 30
16. Click “Run” to run the perfusion program. 20
17. Acquire images using your standard microscope methods. 10
Gradient Formation 0
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Figure 5. Inlet Flow Rate 19. The recommended flow rate for gradient formation is 1‐2 psi. In most cases, it takes about 20 minutes to reach steady state. Once stable, the profile will be linear in the “y‐axis”, with no variation in the “x‐axis.” 20
Mean Diffusion Time (min)
18. Stable spatial gradients are formed by flowing two inlet channels simultaneously. Select the top flow solution (2 or 3) and the bot‐
tom flow solution (4 or 5). The top solution will flow to the top arm of the chamber, and the bottom solution will expose the bottom arm. Solutions will diffuse into the chamber across the perfusion barrier (2 µm pores). A stable diffusion gradient will form in the chamber between the “source” and “sink” channels. Cell Loading: Matrigel Embedded 15
20. Refrigerate the unopened M04G plate at 4°C for ~1 hour. 21. Prepare a cell suspension of 3‐6x106 cells/ml. Place on ice. Dilute 1:8 with chilled Matrigel (final concentration of ~0.5x106 cells/
ml). 10
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22. Repeat steps 5‐9 to load the cell/gel mixture. 0
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Figure 6. Predicted Diffusion Times 1.E‐05
23. Place the plate into a 37°C incubator for 15 minutes to fully po‐
lymerize the gel. The gel will occupy only the culture chamber, leaving the flow channels unblocked. 24. Follow steps 11‐17 for 3D gradient experiments. Note in most cases, molecules will diffuse more slowly through gel than me‐
dium solution. 25. For additional protocols, contact CellASIC. 2544 Barrington Court | Hayward, CA 94545 | www.cellasic.com | 510-785-9846
Operation Tips 26. Flow rates from wells 1, 6, and 8 are ~100X higher than those in figure 5, which applies only to wells 2‐5. 27. For a linear gradient, a flow pressure of 1 psi is recommended; for a sharper transition, faster flow can be used. 28. For temperature sensitive gels such as Matrigel, be careful to keep solutions at 4°C to prevent premature polymerization. 29. The hydrophobic rings in wells 1, 6, and 8 prevent leaking of so‐
lution into these wells during gradient formation. Software Operation Valve on/off Buttons Function Tabs Regulator Setpoints Note: Flow X controls V1 and V2, Flow Y controls V3‐V8 Status Indicator Figure 7. ONIX FG M04 Interface Text Commands: setflow X n ; where n = 0.25‐10 (psi) ; sets flow rate on X (orange) setflow Y n ; where n = 0.25‐10 (psi) ; sets flow rate on Y (blue) open V1 ; V1, V2, …, V8, all ; opens pneumatic valve close V1 ; V1, V2, …, V8, all ; closes pneumatic valve wait n ; n is minutes ; holds current condition until next step end ; ends the program ; shuts off all valves and resets regulators % ; Put at the beginning of line for comments 2544 Barrington Court | Hayward, CA 94545 | www.cellasic.com | 510-785-9846