CytoSoft PDMS Plates of Varying Rigidity
|
Product |
Catalog # |
Starting Volume |
Dilution Factor |
Ending Volume |
Final Working Concentration |
|
PureCol® Type I collagen |
#5005-100ML |
100 mL |
1:30 |
3000 mL |
100 µg/mL |
|
Fibronectin |
#5050-1MG |
2 mL |
1:10 |
20 mL |
50 µg/mL |
|
Vitronectin |
#5051-0.1MG |
0.2 mL |
1:50 |
10 mL |
10 µg/mL |
|
Type IV Collagen |
#5022-5MG |
5 mL |
1:20 |
100 mL |
50 µg/mL |
| Publication | Coating material | Cells |
| Astrogliosis in a Dish: Substrate Stiffness Induces Astrogliosis in Primary Rat Astrocytes | Poly-L-Lysine | Primary Rat Astrocytes |
| Cellular Microenvironment Stiffness Regulates Eicosanoid Production and Signaling Pathways | RatCol Type I Collagen | Human Pulmonary Fibroblasts |
| Deciphering the role of substrate stiffness to enhance internalization efficiency of plasmid DNA in stem cells using lipid-based nanocarriers | High Purity Gelatin A, 1% in PBS | Human Adipose-Derived Stem Cells |
| Dependence of Membrane Tether Strength on Substrate Rigidity Probed by Single-Cell Force Spectroscopy | Not Reported | Human Breast Cancer Cells, Human Cervical Cancer Cells, Human Lung Cancer Cells |
| High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traaction Force Microscopy of Adherent Cells | Fibronectin | Human Umbilical Vein Endothelial Cells |
| Fibroblast Polarization is a Matrix-Rigidity-Dependent Process Controlled by Focal Adhesion Mechanosensing | Fibronectin | Human Foreskin Fibroblasts |
| Matrix Elasticity, Replicative Senescence and DNA Methylation Patterns of Mesenchymal Stem Cells | Not Reported | Mesenchymal Stem Cells |
| Micropatterned Silicone Elastomer Substrates for High Resolution Analysis of Cellular Force Patterns | Fibronectin | Myocytes, Cardiac Fibroblasts |
| Migration of Glial Cells Differentiated from Neurosphere-Forming Neural Stem/Progenitor Cells Depends on the Stiffness of the Chemically Cross-Linked Collagen Gel Substrate | Type I Collagen | Glial Cells, Neural Cells |
| Myosin II Governs Intracellular Pressure and Traction by Distrinct Tropomyosin-Dependent Mechanisms | Not Reported | Primary Human Dermal Fibroblasts |
| Periostin and Matrix Stiffness Combine to Regulate Myofibroblast Differentiation and Fibronectin Synthesis During Palatal Healing | PureCol Type I Collagen | Murine Primary Palatal Fibroblasts |
| Rigid Substrate Induces Esophageal Smooth Muscle Hypertrophy and EoE Fibrotic Gene Expression | Not Reported | Longitudinal Smooth Muscle Cells |
| Rigidity of Silicone Substrates Controls Cell Spreading and Stem Cell Differentiation | PureCol Type I Collagen | Human Mesenchymal Stem Cells |
| Spinal Cord Injury Results in Chronic Mechanical Stiffening | Poly-D-Lysine | Neural Progenitor Cells, Astrocytes |
| Stiffness of the Extracellular Matrix: A Regulator of Prostaglandins in Pulmonary Fibrosis | Not Reported | Human Lung Fibroblasts |
| TGF-B1 Induces Amoeboid-to-Mesenchymal Transition of CD44 Oral Squamous Cell Carcinoma Cells via MiR-422a Downregulation Through ERK Activation and Cofilin-1 Phosphorylation | Laminin-332 | Human Oral Squamous Cell Carcinoma Cells |
| TGF-B-Induced Activation of Conjunctival Fibroblasts is Modulated by FGF-2 and Substratum Stiffness | PureCol Type I Collagen |
Human Conjunctival Fibroblasts |
| Alpha-Parvin Defines a Specific Integrin Adhesome to Maintain the Glomerular Filtration Barrier | Type IV Collagen |
Podocytes |
| L-Plastin Enhances NLRP3 Inflammasome Assembly and Bleomycin-Induced Lung Fibrosis | Collagen I |
Alveolar Macrophages |
|
The Transcription Factor PREP1 Regulates Nuclear Stiffness, the Expression of LINC Complex Proteins and Mechanotransduction |
Not Reported |
HeLa and Mesenchymal Stromal Cells |
|
Substrate Stiffness Induces Neutrophil Extracellular Trap (NET) Formation Through Focal Adhesion Kinase Activation |
Peptite-2000 (RGD), Collagen, Fibronectin, Laminin |
Neutraphils |
We recommend a 60X objective for the imaging plates.
We actually use the method outlined in this journal:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025534
To summarize, we have a layer of gold particles on the bottom of a specially prepared slide. We have the silicone on top of those gold particles, and then we add more gold particles on top of the silicone. We then centrifuge the sample at various speeds and measure the relative displacement/movement of the top beads (on the silicone), compared to the bottom beads (attached to the glass bottom). We then use a formula to calculate the youngs modulus of the silicone for that particular sample.
Using prewarmed media will decrease gas solubility and help prevent bubbles on the surface of the silicone.
The silicone gel can crack if the surface becomes dry.
No. Cell matrix proteins are attached to the surface of the gel via covalent bonding. It is difficult to form a new ECM layer after cell detachment. There are no longer any reactive groups on the surface of the gel after the initial cell culture.
The change in the refractive index of the silicone distorts things a little, so DIC will be possible but not perfect. Also, DIC is only possible on the glass bottom imaging plates, not the 6-well plastic plates.
1.41
The two main issues are:
1. Ineffecient coating of the matrix proteins due to low pH of the coating solution.
2. Insufficient wash of leftover matrix after the coating procedure.
3. After coating, plates needed to be allowed to incubate longer for improved ECM attachment.
We have mostly tested CytoSoft with ECM proteins such as collagen or fibronectin, but a few publications have recently come out using Poly-D-lysine and Poly-L-Lysine instead (while following the rest of the protocol as is).
The surface is decorated with anhydride functional groups.
Plasma use is not recommended. Plasma will induce formation of a hard crust on the surface and will change the mechanical properties of the CytoSoft® products.
Do not freeze the CytoSoft® products.
When frozen, there is a good chance that the silicone surface gets hydrolyzed and absorbs moisture, which would inactivate the binding sites and make the product not-usable.
If you froze the product and it is still frozen, warm the CytoSoft® up at 60C with the bag vented. That will minimize the chance of them absorbing moisture – but there is still a chance that they will no longer be functional.
Using gelatin for coating CytoSoft is often problematic because gelatin often has low molecular weight impurities that block binding sites on the activated surface of the silicone. We recommend using highly purified ECM's instead.
Fibroblast cells secrete their own ECM. The Silicone surface is not efficient for passive absorption of ECM and this is why we use covalent bonding to attach the ECM molecules to its surface for the initial coating.
Fibroblasts use the attached ECM molecules (from the initial coating) to attach and spread on the plate. Fibroblasts are very good at laying down new ECM and after prolonged culture they build a thick layer of ECM on the top of silicone surface.
The problem is that the cell-secreted ECM is not sticking very well to the silicone gel surface and is easy to be pulled by cellular forces. One way to prevent this from happening is to put some sort of 3D hydrogel on top of the cells after they attach so that the hydrogel absorbs or incorporates some of that secreted ECM. A possible downside would be that now the cells are attached to one stiffness (the silicone) but then surrounded by another (the hydrogel).
Click the links below to download the exact plate dimensions and specifications:
No. The humidity in the incubator will deactivate the binding sites on the silicone surface.
The silicone gel will absorb some of this dye. The absorption will be minimal when using the imaging plates.
For the most part, the dye will work just fine. To compensate for absorption, a higher concentration of dye than you would use on plastic may need to be used.
Lipid stains work on Imaging Plates but not on the plastic plates (ie. 6-well). The background stain will be very heavy for the plastic plates because too much dye binds to the thicker PDMS coating. The ~20 micron thickness PDMS on the imaging plates seems to not bind as much dye, allowing you to use the stains.
Yes - you can add Trizol directly to the plates.
The glass bottom for the Imaging plates is #1.5 cover glass thickness, and the layer of silicone coating the glass is thin enough to allow for high resolution imaging. Add oil to objective lens and place the Imaging plate close to the objective, per standard procedure.
Most standard assays performed on cultureware can be performed on CytoSoft plates. This publication may be a good reference:
