We study pulmonary artery endothelial cells and use Matrigel quite a lot. There are several different kind of Matrigel and which one to choose is up to your design. I have read in some article diluting Matrigel with either culture medium or PBS (1:10) but personally never tried it. I have tried 1:1 dilution and it worked well. You keep it on ice when you load it into your culture flask, or plate then put into 37C incubator for at least 30 minutes to solidify it. But you do not allow it to dry-up (use it within a few hours when humidity is appropriate in the incubator). You can also mix you cells of interest with the Matrigel before you plate them. But, the cells need to mix with 4C Matrigel and I do not iknow whether your cells will be viable or not (this is to study the 3D interaction). For endothelial cells some group use home-made rat-tail collagen which is cheap and several good papers used it. I have no clue whether your neuron will be happy or not.

For DRG co-cultures, I used a 1:40 dilution in culture medium (everything should be kept on ice, and don´t let it dry). In this condition, cells are growing properly and are able to mature. I think that you should adjust your own dilution for your purpose. Unfortunately I don´t know any other cheaper alternative.

Good luck Gontzal!

Hi, I've used matrigel to study 3D formations of mammary epithelial cells. Firstly, try not to subject the Matrigel aliquot to so many Freeze/Thaw cycles. And keep it on ice when its not in the freezer or refrigerator. To thaw it, do so hours in advance the same day or overnight in the 4C. Also, keep the plates you intend to plate Matrigel onto in the refrigerator and also on ice, in the hood; just place some aluminum foil or plastic film over the ice to separate ice from getting into your plate/slide. Furthermore, keep a new box of tips that you will be using to pipette the Matrigel, until the very last second before you actually dispense it. For diluting the Matrigel into medium, the medium itself must also be ice cold, so aliquot into tube and keep on ice before diluting Matrigel. Also if you're using Matrigel for differentiation you may want to check if you wanted to use Growth factor reduced (GFR) or the standard mixture with higher levels of growth factors. In using GFR Matrigel you can have tighter control of growth factors that push the cells towards differentiation by adding it to media. I hope this helps.

Best,

Hien

Hynek Wichterle in Neuron, 1996, has a protocol.

1. Thaw Matrigel aliquot SLOWLY. It is best to freeze-thaw the tube several times over the course of a few days to liquefy the material. Put in ice bucket one day, then freeze overnight, then put in back on ice, repeat.

2. Prepare some P200 tips by cutting off the tips to make the opening about 1mm. Do this in the hood with EtOH cleaned razors.

3. Put these tips into a rack, then put the rack into the -20oC.

4. Dissect SVZ tissue in L15 medium and cut into little 100 um sized cubes.

5. Resuspend SVZ cubes (10-20) in about 50 ul of Neurobasal media with B27. Put on ice.

6. Place glass bottomed 35 mm dishes (Hynek dishes) onto ice.

7. Use the -20oC cooled pipet tip to transfer 50ul of Matrigel to the tube with the explants. Mix by pipeting up and down several times, careful to not introduce bubbles.

8. Quickly transfer the mixture to the chilled glass bottomed plate.

9. Let the tissue settle to the bottom of the dish. This puts the explants into the same focal plane. The Matrigel won’t gel until it warms up, so you can let it sit for a few minutes. During this time, you can also move the tissue around with micro forceps to distribute them evenly across the surface.

10. Transfer the plate to the incubator, and let the Matrigel solidify for about 30 min.

11. Add 2 mL of NB/B27 media to the plate. Do this gently so as not to disturb the “plug” of Matrigel.

12. Cells should migrate from the explant overnight.

Hi

I have worked on neural different ion via Matrigel in 1.5 % dilution in medium.

At the first, you transfer matrigel to refrigerator (4^C) overnight,

Prepare 1% matrigel in medium; all of the stages is done on ice.

add 100 microliter of 1% matrigel to 24 well plate and incubate in refrigerator for 15 min, then transfer it to incubator (37^c) for 1 h.

This way you sure that cells will encapsulated in 3D cell culture and will not sense petri dish. when matrigel incubate in 4^c and then transfer to 37^c, at the first forms a homogenous layer of matrigel in the plate and then at 37^c, nanofiber will form.

Then, immediately mix cells with 1.5 % matrigel and add to coated plate (300 microliter) and transfer to incubator overnight. Please note that every 2 min, cells resuspense with matrigel (pipetage). Cells are heavy and maybe reside and if you do not resuspense, cell will not homogenously add to wells and the number of cells will be more in some wells. After overnight, change half medium with fresh medium. Plz every 3 days change 2/5 of medium with fresh media or discard 2/5 of medium and add 1/2 of fresh medium to wells.

If you discard more volume, you will lose some growth factors and necessary factors that cells release for differentiation and survival. plz do not lose them.

Have a nice research

Hi,

we are going to inject cancerous cell lines mixed with matrigel, the high concentration of Corning Matrigel Matrix (20 mg/ml) was uesd, for the first time we diluted the matrigel 1:1 with PBS, but no significant changes observe in the size of tumor.

Anybody know what is the best concentration of the matrigel for subcutaneously injection?

I agree with Shima, with little bit improvement in Matrigel concentration from 1 to 2% makes almost sure of plate/well coating homogeneously and cell suspension with 2% Matrigel (final concentration) in desired media would be a good approach. I think, but not tested, 1.5% and 2% Matrigel doesn't have much effect on cell's activity and behavior. 

https://trevigen.com/product-category/physiologic-cell-culture/extracellular-matrix-proteins/physiologic-cell-culture-extracellular-matrix-proteins-cultrex-bme/?gclid=Cj0KCQiA4bzSBRDOARIsAHJ1UO4FcEsuJi5B1m_0MIDFs2HwB3xiQnG7W-s_UTe9NDTF0UA0N8EaIkAaAmCwEALw_wcB.

In our lab we use this alternative to matrigel, it is called Cultrex, it works great and it is provided in different flavors depending of the use you are intended to do with it. There is a version for xenotransplantation and one for stem cells cultures. it is more robust, less variation lot to lot compared to matrigel and it is cheaper too.

Good luck with your research

Zahra Saberi hello, did you find the solution to your issue ? i'm interested in your question because i'm about to inoculate K7M2 cell lines into mammary fat pad in BALB/C and we want to dilute the suspension in matrigel, but i've never worked with matrigel before an i don't know how to do it ? i thought about inoculating 100.000 cells per 50µl of matrigel , is it doable ? If you have any tips on how to do so please help