I heard that matrigel is used to study matrix degradation in cancer cell invasion assays. What is matrigel?

Does anyone have a protocol for the Invasion assay?

Becton Dickson sells Matrigel. To quote from their website...
"BD Matrigel Matrix is a solubulized basement membrane preparation extracted from EHS mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin 1. At room temperature, BD Matrigel Matrix polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane. Cells behave as they do in vivo when they are cultured on BD Matrigel Matrix. It provides a physiologically relevant environment for studies of cell morphology, biochemical function, migration or invasion, and gene expression."

You may want to consult their website for further information
BD Matrigel Information

I hope this helps.

Edited to fix url.

Have you managed to get hold of a protocol yet? I worked in an institute where one of the groups did invasion assays quite frequently. I can put you in touch if you'd like.Just drop me a private message.

I found this on www.protocol-online.org:

Matrigel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression.


Procedure
1. Thaw Matrigel at 4C overnight.
2. Dilute Matrigel (5mg/ml to 1 mg/ml) in serum free-cold cell culture media (RPMI1640, EMEM, DMEM, etc).
3. Put 100 ul of the diluted matrigel into upper chamber of 24-well transwell
4. Incubate the transwell at 37C at least 4 to 5 h for gelling.
5. Harvest cells from tissue culture flasks by Trypsin/EDTA.
6. Wash the cells 3 times with culture media (RPMI1640, EMEM, DMEM etc)containing 1 % FBS.
7. Resuspend the cells in media containing 1% FBS at a density of 10^6 cells/ml.
8. Gently wash gelled matrigel with warmed serum free-culture media.
9. Put 100 ul of the cell suspension onto the matrigel.
10. lower chamber of the transwell is filled with 600 ul of culture media containing 5 ug/ml fibronectin, as an adhesive subtrate.
11. Incubate at 37C for 20 to 24 h.
12. Remove transwells from 24-well plates and stained with Diff-Quick solution.
13. Scrape off noninvaded cells on the top of the transwell with a cotton swab.
14. Count invaded cells under a light microscope.

Recipes
- Matrigel (Becton-dickinson)
- 24-transwell (Coster)
- Fibronectin(Sigma)
- Diff-Quick staining solution (Fischer Scientific)

labrat said:

Have you managed to get hold of a protocol yet? I worked in an institute where one of the groups did invasion assays quite frequently. I can put you in touch if you'd like.Just drop me a private message.

I am also looking for a protocol. Unfortunatly BD does not have "Guidlines" for the Matrigel.

Sandy said:

I heard that matrigel is used to study matrix degradation in cancer cell invasion assays. What is matrigel? Does anyone have a protocol for the Invasion assay?

Matrigel is a basement membrane matrix composed of extracellular matrix proteins from mouse tumor. It contains some growth factors and other components that allow cells, i.e. neurons, to regenerate. I use it plate neurons for cell culture. The matrix can affect gene expression is some cells as well. Be careful with the matrigel because it is highly sensitive to warmer temperatures. The efficiency of the matrigel is highly dependent on temperature.

Chemicon has a protocol for the Invasion assay. Check out the website: www.chemicon.com/Resource/litlibrary/476CellInvasion.pdf

homefroggy7 said:

Sandy said: I heard that matrigel is used to study matrix degradation in cancer cell invasion assays. What is matrigel? Does anyone have a protocol for the Invasion assay? Matrigel is a basement membrane matrix composed of extracellular matrix proteins from mouse tumor. It contains some growth factors and other components that allow cells, i.e. neurons, to regenerate. I use it plate neurons for cell culture. The matrix can affect gene expression is some cells as well. Be careful with the matrigel because it is highly sensitive to warmer temperatures. The efficiency of the matrigel is highly dependent on temperature. Chemicon has a protocol for the Invasion assay. Check out the website: www.chemicon.com/Resource/litlibrary/476CellInvasion.pdf

Thank you so much for this valuable information, Does anyone have a list of companies that sale Matrigel?

BD Biosciences does:
Link

Sigma has one:
http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/E1270

But how do you use the Matrigel for 3D cell cultures. How do you handle it. Like, how do you make it liquid to make transfer into wells possible??

fisher said:

But how do you use the Matrigel for 3D cell cultures. How do you handle it. Like, how do you make it liquid to make transfer into wells possible??

From BD's Matrigel datasheet:

MATRIGEL BASEMENT MEMBRANE MATRIX WILL GEL RAPIDLY AT 22oC TO 35oC. THAW
AT 2-8oC OVERNIGHT ON ICE (MATRIGEL MAY GEL AT SLIGHTLY ELEVATED TEMPERATURES IN A
REFRIGERATOR), KEEP PRODUCT ON ICE BEFORE USE. USE PRE-COOLED PIPETTES, PLATES AND
TUBES WHEN PREPARING MATRIGEL BASEMENT MEMBRANE MATRIX FOR USE. GELLED
MATRIGEL MAY BE RE-LIQUIFIED IF PLACED AT 2-8°C ON ICE FOR 24-48 HOURS.

Therefore, matrigel is sold as frozen. When you thaw it slowly on ice, it becomes liquid. If you allow it to warm up, it will gel. From the gel state it should be possible to make it liquid again by placing Matrigel at 2-8 C on ice for 24-48 hours.

Hi,

I bought the BD Matrigel # 356231. I want to grow my cells in 3D resuspension. However, I cannot find any information about the concentration of this product and how much I can dilute it with my culture medium and it will still form 3D-gel. Do you know? Have you tried this?
Thank you very much!
x

millipede said:

fisher said: But how do you use the Matrigel for 3D cell cultures. How do you handle it. Like, how do you make it liquid to make transfer into wells possible?? From BD's Matrigel datasheet: MATRIGEL BASEMENT MEMBRANE MATRIX WILL GEL RAPIDLY AT 22oC TO 35oC. THAW AT 2-8oC OVERNIGHT ON ICE (MATRIGEL MAY GEL AT SLIGHTLY ELEVATED TEMPERATURES IN A REFRIGERATOR), KEEP PRODUCT ON ICE BEFORE USE. USE PRE-COOLED PIPETTES, PLATES AND TUBES WHEN PREPARING MATRIGEL BASEMENT MEMBRANE MATRIX FOR USE. GELLED MATRIGEL MAY BE RE-LIQUIFIED IF PLACED AT 2-8°C ON ICE FOR 24-48 HOURS. Therefore, matrigel is sold as frozen. When you thaw it slowly on ice, it becomes liquid. If you allow it to warm up, it will gel. From the gel state it should be possible to make it liquid again by placing Matrigel at 2-8 C on ice for 24-48 hours.

The concentration differs from batch to batch. You have to call BD and give them the lot # to get the concentration of a particular batch.

Here's a paper with thorough description of the method for cell culture in 3D matrigel.

Methods
Volume 30, Issue 3 , July 2003, Pages 256-268

This is the pubmed link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12798140&query_hl=8

Good luck!

[quote=xzhao]Hi,

I bought the BD Matrigel # 356231. I want to grow my cells in 3D resuspension. However, I cannot find any information about the concentration of this product and how much I can dilute it with my culture medium and it will still form 3D-gel. Do you know? Have you tried this?
Thank you very much!
x

Does anyone know the approximate concentration of matrigel as sold by BD. Neither Sigma nor BD will tell me, and it's pretty hard to design an experiment without a concentration.


[quote=shard602]The concentration differs from batch to batch. You have to call BD and give them the lot # to get the concentration of a particular batch.

Here's a paper with thorough description of the method for cell culture in 3D matrigel.

Methods
Volume 30, Issue 3 , July 2003, Pages 256-268

This is the pubmed link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12798140&query_hl=8

Good luck!

[quote=xzhao]Hi,

I bought the BD Matrigel # 356231. I want to grow my cells in 3D resuspension. However, I cannot find any information about the concentration of this product and how much I can dilute it with my culture medium and it will still form 3D-gel. Do you know? Have you tried this?
Thank you very much!
x

[/quote]

does anyone know the complete list of components in Matrigel? does it have glucose in it or is that only introduced if you mix it with growth medium? Thanks!

chemist27 said:

does anyone know the complete list of components in Matrigel?

Matrigel is a natural product. It's make up is not compeletly defined, so you are not going to be able to obtain an absolutely complete list of components. From the product datasheet:

Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin

There are different grades of Matrigel which have been cleaned/purified to various degrees.

chemist27 said:

does it have glucose in it or is that only introduced if you mix it with growth medium? Thanks!

Essientially no Glucose, you have to add that.

Google Scholar Link to Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan... by Kleinman describing the isolation of a Matrigel like material (As referenced by BD Bioscience who produce Matrigel).