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Once you have found the optimal size and optimal pattern for your assays, your cells will not suffer from micropatterning. They will go through their cell division cycle at the usual pace.
To discover which pattern is the best for you, you can use our Starter CYTOOchip. We can also provide you Custom micropattern designs to meet your exact requirements, for instance for cells who need cell-cell contacts.
Replace your coverslip or your glass bottom microplate with a CYTOOchip or a CYTOOplate and carry out your usual cell assay with minor modifications. Fix or video record during the first cell division cycle. Use your usual drugs, labels and probes.
Use our Starter's CYTOOchip which offers 4 standard micropattern geometries in 3 sizes. Perform your cell assay on the Starter's CYTOOchip and analyze which micropattern works best. Description of micropatterns and uses may also bring useful information
Cells do not spread out on the micropatterns. They keep a round shape and are bright in phase contrast. Cells may also make blebs
Cells adopt various shapes on a given geometry. In time lapse microscopy, cells never seem to stabilize on the micropatterns but rather move back and forth. This induces a large variability in cell phenotypes.
Our products are always submitted to quality controls, but if you notice scratches on your chips, please check the following points:
CYTOOchips must be grasped by the edges with tweezers. We recommend you not to touch the center part of the CYTOOchip, which may destroy locally the cytophobic surface.
For mounting the chips onglass,always use a mounting medium. Take care to keep the chip wet during your experimentation.
Change gasket (spare gaskets can be bought from CYTOO)
Never use oil (oil makes the gasket swell and provokes leaks)
Check that your CYTOOchambers have not been stored near a strong magnetic field, which may demagnetize them.
Check that at least 3 ml was added to the well/dish, and be careful not to rock or swirl the dish, because your cells may concentrate at the center of the chip. Let your cells sediment under the hood for 10 min before moving them to the incubator.
Check that at least 100 μl was added to the well. With less volume, a meniscus effect leads to heterogeneous cell sedimentation, so a higher volume may be necessary to compensate for dead volumes of dispensing instruments or reservoirs. For the same reason, avoid rocking or swirling the dish.
Make sure to keep cells under the hood for 15-45 minutes before moving them to the incubator.
Maintain the chip at the bottom of the well by pressing on the CYTOO logo with the tip of a pipette.
Wait longer. Some cell types take more than two hours to start spreading on micropatterns. In this case try lower cell density to avoid cell aggregation.
Are you working with the right adhesion protein for your cell line? You can try coating a different protein on our activated CYTOOchips. We provide a set of recommended protocols for coating the most widely used proteins (collagen, laminin, polylysine, etc)
Fibronectin micropatterns and CYTOOchambers are not toxic in any way for your cells if correct culture conditions are provided. This problem can have various environmental origins:
Please make sure that you followed correctly the procedure to use the CYTOO products, notably regarding the sufficient volume of culture medium.
Contamination by bacteria, yeasts or fungi. Check their presence and decontaminate CYTOOchambers using ethanol 70°. Be sure to work under sterile conditions.
The temperature, moisture or CO2 conditions are not correctly supplied by your incubator.
Cells are not cultured in the correct culture medium. You can also add Hepes buffer pH = 7.4 in the medium for long term experiments on micropatterns.
Cells are cultured with selection pressure antibiotics, which can stress the cells if concentrations are not adjusted.
Cells are stressed because they stayed for a long time in trypsin/EDTA solution before seeding on micropatterns. Try to dilute trypsin/EDTA solution in Hepes containing culture medium to remove your cells from dishes before their seeding on micropatterns.
Cells suffer from freezing and thawing processes. After cell thawing, imperatively culture them during one week in classic conditions before starting experiments on micropatterns.
Check cell density after trypsinization. Try higher numbers of cells per well and/or wait longer before changing the cell medium. Your cells may need a little longer to attach. Monitor the first signs of spreading under the microscope and wash then. You may also need to wash more gently depending on the cell type.
Check cell density after trypsinization. Try lower numbers of cells per well and/or reduce the time between initial seeding and changing the cell medium. Your cells may be attaching very quickly and floating cells are attaching to patterns that were already occupied. Monitor the first signs of spreading under the microscope and wash then. You may also need to wash more strongly depending on the cell type.
This situation is often encountered with cells that tend to clump together (epithelial cells for example). The idea is to try to favor adhesion to the fibronectin (through integrins), versus adhesion between cells (through cadherins).
Here are a few ideas:
Take great care in dispersing as well as possible the initial cell suspension by pipetting up and down many times.
Using a medium depleted in calcium (with 300 μM EGTA) while seeding would probably help limit cell aggregation (through cadherins which are calcium-dependent) before adhesion to the surface (through integrins).
All media and dishes should be preheated to 37° to help adhesion start immediately (cell clumping is less temperature dependent).
Using a diluted cell suspension would also help (less chance of them meeting before they fall to the surface), but of course less micropatterns will be occupied.
Treating the cell suspension with Accumax (Millipore) before seeding may also be efficient in avoiding cell clumping.
Check the cells that are on the fully adhesive area of the chip. Cells should spread out on this area as in a coated culture dish. If not, control cell viability.
The adhesive protein provided might not be the appropriate protein for your cell type. Contact us, we can offer Ready-to-Coat micropatterns that can be used to coat your own adhesive proteins, or micropatterns with a
Patterns are too large, which results in cells moving from one side to the other. This induces a large variability in cell shape. Try smaller sized patterns.
Our products must be refrigerated at 4°C, under dry conditions.
Do not freeze at any time your plates or the glass bottom may become detached from the upper frame
Use our products before the expiry date indicated on the blister and on the bag. Once the bag is opened, use your chips and plates rapidly as optimal storage conditions are no longer guaranteed.
Be careful not to store your CYTOOchambers near a strong magnetic field (solenoid, high content screening system…), otherwise the magnets may be demagnetized, and your CYTOOchambers may leak.
The CYTOOchambers can be autoclaved, but as a precaution, do not heat them over 120°C.
