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ECM Gel Cell Attachment Protocol

ECM Gel: Cat. No. E1270

  • Thaw gel overnight at 2-8°C before use.
  • Dispense gel to wells of a multiwell plate using pipettes, plates, etc. pre-cooled to 2-8°C. (For a 96 well plate, use 50-100 mL/well). *ECM gel also may be diluted up to twofold with cold (2-8°C) Dulbecco’s Modified Eagle’s Medium. Dilution may help in cases in which the product is still gelatinous in the cold. Gel dilutions should be made before it is added to the plate. *ECM gel will gel within 5 minutes at 20°C. For prolonged manipulations, work should be conducted below 10°C.
  • Cells may then be plated on top of a thin gel layer (0.5 mm) or cultured inside a 1 mm layer. In the latter application, cells should be added to the gel prior to plating at a recommended density of 3-4 x 104 cells per ml.

ECM Gel: Cat. No. E6909

  • Thaw gel overnight at 2–8 °C before use (The gel may contain precipitates which do not influence its activity).
  • Dispense the gel into wells of a multiwell plate using pre-cooled devices. For a 96 well plate, dispense 50–100 L/well. The ECM Gel, Growth Factor Reduced will gel within 5 minutes at 20 C. For prolonged manipulations, work should be conducted below 10°C.
  • Cells may be plated on top of a thin gel layer (0.5 mm) or cultured inside a 1 mm layer. In the latter application, cells should be added to the gel prior to plating at a recommended density of 3–4 x 104 cells per ml.

*To dissociate cells from the gel, use protease (dispase) in PBS without calcium, magnesium, and EDTA, at a concentration of 0.6-2.4 units

Ask a Scientist!

Section 1: ECM gels characteristics:

Is this derived from cell lysate or actual cell matrix?
The ECM gel is produced from whole tumors (not separated cells). It is a result of a total extraction of the tumors.

Is this the same as Matrigel™ (from BD)?
ECM gel is the equivalent of Matrigel™ from BD, only produced by Sigma-Aldrich.

Are there any attachment factors added in addition?
Product E1270 is extracted from Engelbreth-Holm-Swarm mouse sarcomas  with no external additives.

The main components of the basement membranes are laminin, collagen IV, entactin, and heparan sulfate proteoglycan (HSPG).  The first three are usually present approximately in equimolar amounts, HSPG in a lower quantity, but as this is an extraction from a biological source, we cannot state fixed values of relative component concentrations.

What is the calcium concentration in ECM gel?
The approximate concentration of CaCl2 in the ECM solution is 0.265 mg/mL.  per mL.

Is there antibiotic  like gentamicin?
Our ECM gel (E1270) contains Gentamycin.

Is it possible to purchase E1270 in DMEM without Na Pyr.?
The DMEM used in the preparation of E1270 does not contain Sodium Pyruvate.

We safely assume that no Na Pyruvate is added to starting material during manufacturing process.

Is the presence of mouse retrovirus (MLV and relatives) is tested?
We do not test product E1270 for virus presence. However, several product lots were tested in the past and found free of LDEV, and our raw material supplier Harlan added the LDEV test to their SPF tests list thus the source (mice) are cleaned for this virus.Moreover, by risk assessment, RNA viruses should not survive the manufacturing procedure. Thus, we assume the product is LDEV free.

Section 2: ECM gels use guidelines:

Is it suitable for 3D cell culture if frozen again and re-thawed next time?
We would not recommend thawing and then freezing the product E1270, ECM gel from Engelbreth-Holm-Swarm murine sarcoma.  Please note that once it is thawed it can not be refrozen.What I can suggest the customer do is make up gels and store them at 2-8 C.  You can store them properly covered for a while or till the time they start cracking up.

The data sheet for E1270 says that it may be diluted up to twofold. What does that mean?
ECM gel can be diluted twofold with cold Dulbecco’s Modified Eagle’s Medium (DME). This means 1 part ECM gel and 1 part DME.

Do you have data about gel degradation in presence of cells?
We know that product does not degrade after 7 days of co-incubation with cells.

How can I retrieve my cells from the ECM gel?
Dispase (Cat. No. D4818) is a Bacillus-derived neutral protease that is recommended for recovering cells cultured on ECM (Cat. No. E1270). Dispase will yield a single cell suspension far more effectively than trypsin, collagenase, or other proteolytic enzymes. Dispase is gentle and will not damage cells harvested for sub-cultivation or other study.

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