Matrigel

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Sandy
Sandy's picture
Matrigel

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?

montgomj
montgomj's picture
Becton Dickson sells Matrigel

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

Edited to fix url.

I hope this helps.

You can also check this protocol on the is site for plating in matrigel. Edited to add protocol.

Matrigel plating protocol

labrat
labrat's picture
Have you managed to get hold

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.

vanishing
vanishing's picture
I found this on www.protocol

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)

fisher
fisher's picture
labrat wrote:Have you managed

labrat wrote:

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.

homefroggy7
homefroggy7's picture
Sandy wrote:I heard that

Sandy wrote:

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

DD
DD's picture
homefroggy7 wrote:Sandy wrote

homefroggy7 wrote:

Sandy wrote:
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?

vanishing
vanishing's picture
BD Biosciences does:Link
fisher
fisher's picture
But how do you use the

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??

millipede
millipede's picture
fisher wrote:But how do you

fisher wrote:

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.

xzhao
xzhao's picture
Hi,

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 wrote:

fisher wrote:
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.

shard602
shard602's picture
The concentration differs

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!

xzhao wrote:

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

Erin
Erin's picture
Does anyone know the

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.

shard602 wrote:

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!

xzhao wrote:

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

chemist27
chemist27's picture
does anyone know the complete

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!

Richard Taylor
Richard Taylor's picture
chemist27 wrote:does anyone

chemist27 wrote:

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:
Quote:

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 wrote:

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).

Chiara
Chiara's picture
Hi,

Hi,
the protocol for cell invasion assay from www.protocol-online.org seems affordable, but does anyone know if is available a staining solution cheaper?
Thank you

Richard Taylor
Richard Taylor's picture
Are you refering to the "Diff

Are you refering to the "Diff Quick" stain?

In which case, it's just a proparity brand name for :
Diff-Quick fixative (1.8 mg/L triarylmethanein methyl-alcohol)
Diff-Quick solution 1 (1 g/L xanthene in sodium acide-preserved buffer)
Diff-Quick solution 2 (0.625 g/L azure A and 0.625 g/L methylene blue in buffer)

What are you doing?
Any reason not to keep it simple and fix with 70% ethanol and stain using 1% methylene blue
as: http://www.nature.com/bjc/journal/v89/n11/full/6601429a.html ?

Chiara
Chiara's picture
I have to find out if my

I have to find out if my cells are able to invade the extracellular matrix as step in tumor metastasis.
I never done this sort of investigation and initially I found different kinds of kit suitable for this purpose (chemicon, calbiochem, etc...).
So, I need to recollect my cells after their passage throu the membrane and stain to count them. The protocol that you suggest could be appropriate.
Thank you.

edanto
edanto's picture
Quote:Its major component is

Quote:

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

Does anyone know what cellular enzymes degrade the matrigel and help the cells to pass through it?

A quick pubmed pointed me at plasmin, gelatinase and some of the MMPs - but many of the papers were quite old. Help with newer refs, anyone?

many thanks
e

Jo
Jo's picture
We've used Matrigel for

We've used Matrigel for invasion assays. It's a matrix you can coat onto culture well inserts then seed your cells ontop of, then monitor their invasion through the Matrigel into the bottom of the well by confocal microscopy, or by counting the number of cells that invade right through. Sorry I don't know where our protocol is.

Jo
Jo's picture
edanto wrote:Quote:Its major

edanto wrote:

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

Does anyone know what cellular enzymes degrade the matrigel and help the cells to pass through it?

A quick pubmed pointed me at plasmin, gelatinase and some of the MMPs - but many of the papers were quite old. Help with newer refs, anyone?

many thanks
e

It depends what cells you're looking at. The major gelatinases in the cells we use are MMP-2 and MMP-9. If you do a pubmed search it should be easy to find a review on them.

Seagull
Seagull's picture
fisher wrote:But how do you

fisher wrote:

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??

Here it introduces the 3D Assay in Matrigel:
http://www.lbl.gov/lifesciences/BissellLab/labprotocols/ehs3dassay.htm

Tony Rook
Tony Rook's picture
Here is a link to the

Here is a link to the following protocol within the Cell Biology protocol section:

Matrigel Aliquoting and Plating

SeekerOfScience
SeekerOfScience's picture
Has anyone heard of a product

Has anyone heard of a product called BioNuesis (BioNuesis.com) put out by Histogen Inc. They are selling the product for $185 but are currently giving away samples for anyone to experiment with. Let me know if anyone knows anything about the product.

ThomasH
ThomasH's picture
SeekerOfScience wrote:Has

SeekerOfScience wrote:

Has anyone heard of a product called BioNuesis (BioNuesis.com) put out by Histogen Inc. They are selling the product for $185 but are currently giving away samples for anyone to experiment with. Let me know if anyone knows anything about the product.

BioNuesis™, stem cell culturing kit, developed by Histogen, Inc., utilizes soluble human extracellular matrix (ECM). It is a human alternative to MatriGel™. BioNuesis contains no tumor growth factors and is manufactured from neonatal fibroblasts. Given this novel manufacturing process and by instituting quality control testing, BioNuesis provides a uniform ECM that mouse tumors cannot.

This soluble human feeder-layer ECM contains proteins that provide an entirely human source of the ECM molecules laminin, fibronectin and collagen type IV, which have been shown to support human embryonic stem cell expansion.
BioNuesis is in a ready-to-use format for cell culture applications. BioNuesis has been formulated to be used for research only, and not for therapeutic or diagnostic purposes.

For additional information, visit the BioNuesis website at bionuesis.com.

minylim
minylim's picture
Drop me personal message if

Drop me personal message if you need the protocol for matrigel invasion assay.

spchaki
spchaki's picture
Hi

Hi
I am using BD matrigel , cat. no 354263 of lot no. A6520 and the concentration is 9.2 mg/ml. It varies with lot no. and BD may be having range from 7.5-9.5 mg/ ml. You can confirm calling them. Good luck.
Sankar

happy life
happy life's picture
hello  Now  I plan order the

hello  Now  I plan order the BD matrigel ,would you please tell me what is different from the high concentration BD matrigel  and the normal . There are many kinds of BD matrigel  , I do not  know which is better for the cell invasion assay ?

manderson
manderson's picture
High concentration Matrigel

High concentration Matrigel is the one most suitable for in-vivo implants.  I have made extensive use of it in mice this last year and am very pleased with some of the results I am getting

I am sure others who use this product in-vitro will confirm that the standard concentration is more likely to suit your need and be more economical

happy life
happy life's picture
Thank you so much for your

Thank you so much for your timely reply

happy life
happy life's picture
Thank you !

Thank you !

Does the phenol-red affect the results of stain with Diff-Quick kit .

which is better for cancer cell invasion assay ,phenol-red free or standard BD

matrigel matrix ?

Is the  growth factor reduced BD matrigel matrix better for this assay ?

minylim
minylim's picture
I don't think phenol red

I don't think phenol red affect invasion assay but not sure about Diff-Quick kit. You may want to talk to tech support of the manufacturer.

GFR matrigel is denifitely lot better than regular matrigel matrix for invasion assay. Having more than triplicate for each experiments helps,  you can easily exclude outlier often the results are inconsistent.

happy life
happy life's picture
Thank you so much !

Thank you so much !

paritosh
paritosh's picture
Hello friends

Hello friends
Matrigel is patent of Becton Dickinson
The various forms of matrigel available are:

BD BioCoat™ Matrigel™ Matrix Cellware
 
 
 
 

354432
BIOCOAT OTHER
28
CAS
6-well plates

354503
BIOCOAT OTHER
28
CAS
12-well plates

354433
BIOCOAT OTHER
28
CAS
24-well plates

354508
BIOCOAT OTHER
28
CAS
48-well plates

354460
BIOCOAT OTHER
28
CAS
35 mm culture dishes

 
 
 
 
 

 
 
 
 
BD Matrigel Matrix—Thin Layer

354603
BIOCOAT OTHER
28
CAS
6-well plates

354605
BIOCOAT OTHER
28
CAS
24-well plates

354607
BIOCOAT OTHER
28
CAS
96-well plates

354602
BIOCOAT OTHER
28
CAS
35 mm culture dishes

354601
BIOCOAT OTHER
28
CAS
60 mm culture dishes

354600
BIOCOAT OTHER
28
CAS
100 mm culture dishes

 
 
 
 
 

 
 
 
 
BD Matrigel Matrix for Hepatocytes

354510
BIOCOAT OTHER
28
CAS
6-well plates

354634
BIOCOAT OTHER
28
CAS
100 mm culture dishes

 
 
 
 
 

 
 
 
 
Growth Factor Reduced Matrigel Matrix for Smooth Muscle Cells

354635
BIOCOAT OTHER
28
CAS
24-well plates

BD BioCoat™ Matrigel™ Matrix Cell Culture Inserts
 
 
 
 

354443
BIOCOAT INSERTS
26
CAS
0.4 µm inserts in four 6-well plates

354447
BIOCOAT INSERTS
26
CAS
0.4 µm inserts in two 24-well plates

These are well validated with number of references published:

Anyone interested can also surf

puramatrix
hydromatrix

Regards
Paritosh Shekhar

Yang891015
Yang891015's picture
 Hi, can you send me a copy

 Hi, can you send me a copy of the protocol? Thanks so much!

Vampire squid bubble
Vampire squid bubble's picture
 

 
Matrigel

From Wikipedia, the free encyclopedia
 
 

For citing references in Wikipedia, see Wikipedia:Citing sources.
Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and marketed by BD Biosciences and by Trevigen, Inc., under the name Cultrex BME. This mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for culturing cells.[1][2]

Anwar
Anwar's picture
what is difference between

what is difference between growth factors and foetal calf serum in term of biology, can we use growth factors instead of serum media to achieve optimize growth?