HI Durgesh

I do it pretty much as you explain - ie I make up enough for all my injections (and an extra 1-2 injections for pipetting error). For example, for 6 injections of 100 µL per injection, I make up enough cell suspension for 7 injections (ie 700 µL) to account for pipetting loss (which I find can be an issue with Matrigel).

I first make up 50% GFR Matrigel and 50% ice-cold PBS, mix well. Then I resuspend (without introducing bubbles) a cell pellet with this mix to give an appropriate cell dose per 100 µL and leave this on ice. Pretty soon after making the cell suspension (so as not to allow the cells to settle) I then use a 29G insulin syringe (with no dead space) to draw up and inject 100µL at a time into separate mouse flanks s.c.

I've tried both ways (individual preparations and using a mastermix) and I definitely find the mastermix approach much more reproducible - after all there's so much variability in mouse expts, the last thing you want to do is increase the technical error by making individual cell suspensions in my opinion.

Best of luck with your experiments

Al

I use BD high purity growth factor reduced Matrigel and that usually comes at a concentration of around 10-12 mg/ml protein content. I dilute that 1:1 with PBS to a concentration of about 5-6 mg/ml. Regarding your cells I have used 1 million cells for such experiments. When you have your cell pellet, dilute your Matrigel to the right concentration on ice and resuspend your cells in this solution. Keep cold, otherwise the Matrigel polymerises. I inject 100 ul of cells in this solution subcutaneously.

Dear i am also first time to use this model type and i am in conversation with a Prof. from USA if you need we can contact with each other through a project

Hi. I used the Matrigel some time ago. We added FGF2 to the gel instead of cells and injected 0.5 ml subcu. per mouse. To simplify injections, we had individual 0.5 ml Matrigel samples in tuberculin syringes sitting in ice. After isofluorane anesthesia you have ~20 sec to inject.

Take care to avoid airbubbles in the Matrigel and inject it slowly.

Article Plasminogen Activator Inhibitor-1 Regulates Tumor Growth and...

I use matrigel from BDBiosciences: Basement Membrane Matrix, High Concentration (HC), 10 ml (Catalog Number 354248).

I thawed the vial out in ice box within fridge over night and made aliquos, then store at -20C.

For injection, I dilute the matrigel with FBS free media 1:1 ratio and inject 100ul.

Just follow the manual and instructions, you will be fine. Basically, KEEP EVERYTHING COOL ON ICE (tips, matrigel/cell mixture, syringe, needles). After 1:1 dilution, the matrigel/cell mixture is easy to handle, not that thick...

Hi. I used 2-4 × 10 6 cells that were inoculated into nude mice (5-week-old athymic female BALB/c mice) after resuspension in 200 μL growth factor–depleted Matrigel (Becton Dickinson). I usually perform two injections per mouse, one on each thigh of the animal. Important: keep the mix matrigel plus cells always on ice to avoid polymerization. Tumor mass was monitored every week.

Hi.. I have used matrigel from Sigma. They recommend to make a stock of 10 mg/ml and then use 100 uL of this for cells. We used to re-suspend about 3-5 million cells in 100 uL of matrigel made in serum-free medium and taken on ice before injecting. Remember the matrigel gels within 5-10 mins at any temperature above 20 degrees.

Try this out. I am sure it will work for you. Remember, although matrigel works in vivo, the ability of HuH7 cells to develop into a tumor, which I presume is what you are looking for, is also critical. If these cells cannot form effective tumors, even matrigel cannot be of much help.

Hope this information is of some help to you.

I use matrigel from BDBiosciences: Basement Membrane Matrix, (Catalog Number 354248). Always thaw the vial in ice box within 4 degree refrigerator overnight and make aliquots of the left over matrigel. For injection, I dilute the matrigel (1:1 ratio) in complete media (the one which you use to grow your cells in) and inject 100 ul. I always find that one million cells is an optimal number for a xenograft but you might want to see prior publications to see what others have used and you should use the same number. You should not have any issues with tumors. Make sure you implant then subcutaneously. Take help from someone if you are not familiar with the technique. It is important that the cells are subcutaneous or else you will have tumor infiltrating peritoneal cavity which could kill the mice.

Last thing would be to ALWAYS keep everything (matrigel, cells, or pipette tips for mixing the cells) on ice. And ya mix the cells slowly between injections if you have large numbers of mice to inject. Best of luck~!

I agree with my colleagues. Keep it/everything cold at 4°C and don´t dilute MG higher than 1:3 with cells. Also important: there is the "normal" MG and growth factor-depleted MG. You should consider carefully what the best option for the MG would be in your experimental setting to avoid side effects of certain growth factors that may interfere with your signaling system of interest. Good luck!

reduced matrigel from Fisher, 1:1 ratio, see paper by Carlson and all

Establishment, Maintenance, and in Vitro and in Vivo Applications of primary human Glioblastoma Multiforme GBM Xenograft Models for Translational Biology studies and Drug Discover.

Current Protocols in Pharmacology March 2011

you can use both pure or diluted (1:1 in sterile PBS) Matrigel; this will help tumor take after subcutaneous injection, since Matrigel will gelify at 37°C. In that way, tumors will grow faster when compared to cell injections with PBS alone.

The above answers are great. A few more tips may help:

Please keep in mind that the cell number you use will depend on your cell type. I use matrigel from BD (growth factor reduced) and thaw the vial overnight at 4 degrees. Matrigel gets solidified in room temperature and in -20. I prepare small aliquots in sterile eppendorf tubes, store those in -20 degrees C and take out only the amount needed for each experiment. I thaw the required amount on ice on the day of the experiment. You can prepare 500 ul aliquot, if you are injecting at least 10 mice per experiment. Make sure to resuspend your cells in such a concentration so that the desired number of cells injected in each mouse is suspended in 50 ul media. Add this to the matrigel and mix slowly by pipetting up and down ON ICE. Matrigel also gives bubble quickly, so be gentle. Inject 100 ul of this mixture to each mouse. Mixing the matrigel with cell suspension should be done carefully, as matrigel is viscous and the cell may not mix homogeneously. If injecting a large number of mice, make sure to intermittently mix the suspension to make sure that the cells are not lodged at the bottom. When injected, it will take a few seconds for the matrigel to solidify at the site at mice's body temperature, so wait a few second before pulling the needle out.

Hi, use matrigel at 10 mg/ml , keep on ice and inject very slowly to let it gel at the injection site and not diffuse under the skin.

Here is our protocol

Article Inhibition of angiogenesis in vivo and growth of Kaposi's sa...

If you want to perform an angiogenesis assay, matrigel plug assays have many drawbacks. I recommend you use the "directed in vivo angiogenesis assay" (DIVAA). You can find the protocol in: Martínez A, Zudaire E, Portal-Núñez S, Guédez L, Libutti SK, Stetler-Stevenson WG, Cuttitta F.

Proadrenomedullin N-terminal 20 peptide is a potent angiogenic factor and its inhibition results in reduction of tumor growth.

Cancer Research 64: 6489-6494 (2004).

We resuspend the desired numer of cells to inject in 50 ul PBS and add 50 ul matrigel just before sub-cut injection (obviously scaling up for the number of mice you need plus a generous extra volume).

Be careful while handling Matrigel as it will very easily become gloopy and difficult to pippete and inject. We normally thaw the vial when it arrives and do several 1 ml aliquotes. On the day of injection I move the aliquotes to a fridge and thaw it slowly over a few hours. If the Matrigel warms up close to room temperature it will begin to solidify.

When you use it, remember to put your Matrigel on ice. I used it before, but i don't use it any more, because most of cancer cells can form tumor with Matrigel. Good luck!

I suggest to keep the syringe on ice before to load the cell suspension in matrigel. It helps a bit

We use high concention Matrigel (4 million cells in 50 ul Matrigel + 50 ul medium per mouse). You need to put the Matrigel on ice at least 2 days prior injection and to change the ice once or twice a day

Why are we answering the question without adding anything new and substantial. I would like to ask everyone (myself included) to please read the answers first before adding any input. That would be helpful to the person asking the question as well as others who read the forum.

You are very right, Imtiaz Siddiqui. I think people do that sometimes, just to add to the number of participants in the forum and sometimes, I'm of the thought that it will give the question generator more confident to follow a particular protocol that might have been addressed by a first answer respondent to the question.....................its just my thought.

All the tips have been given above - so I have not much to add, appart from the fact that to my opinion matrigel is not mandatory when performing s.c. xenografts, especially when handling such large amount of cells to be implanted (i.e., one million cells, which seems to be a standard) - matrigel is more helpful when performing orthotopic engraftments, or when you attempt to inject fewer cells to make your experiment last longer (we gave up the "one-million cells paradigm" in my lab, we now try to graft no more than 100 000 cells (with 60% matrigel indeed), that leaves you plenty of time to evaluate drug effects for instance - but I guess it all depends of the very nature of your experiments). Back to s.c. grafts, frankly I did not see a sharp reduction in inter-individual variability in tumor volumes by using matrigel. Just make a try and make your own opinion.

I liked using Cultrex basement membrane from Trevigen because it was not only cheaper but also generally had higher protein content (which my cells liked), but all other considerations remain. Remember that this matrix is generated by a tumor cell line (Engelbreth-Holm-Swarm tumor), so it will contain not only structural components (entactins, collagen IV, laminins) but growth factors as well - even the GFR versions. We ended up not having to use it as our syngeneic mouse lines grew s.c. without (at 0.2 -1e6 cells/animal). You may want to run concurrent cage of "matrigel-free" implants to see if your cells will grow without it, and/or determine the variability in tumor growth with/without the matrix.

I agree with Celine. We use 50/50 cells in medium/matrigel. Again place materiel on ice in fridge 3-4 days prior to use. We keep tips, pipettes and eppendorfs to be used in the fridge also. Keep everything on ice at all times throughout preparations and prior to injection.

Hello Kirsty,

Using matrigel is very tricky, you have to be very careful about temp. Matrigel be at solid state at lower temp. as well as at higher temp. than 4'C.

keep matrigel at 4'C atleast for 12 hrs before using then it will become a viscous liquid.

Now Plan your exp. in a way that single cell suspension(in PBS), needle, syringe should be at 4'C. just before loading cells add matrigel in 1:1 ratio (total volume should be 0.2 ml),mix it uniformly and go for injection.just keep in mind that temp. should be around 4'C during the whole procedure.

Best Luck...

Go to Nature Protocols volume 7, page 1138 by Fridman et al. It has all the tips and uses.

A technical tip with an injection: as both cells and the Matrigel like to bind to the plunger, when I prepare the syringes of Matrigel mixed with cells I do it in a cold room, cells mixed with Matrigel are usually already put on ice in 2.2 ml Eppis (as they have the round bottom), one Eppi per one syringe. Then I take off the thin needle from the insulin syringe, take same air into syringe (around 100 ul) and then take in the mix of Matrigel with cells (it is good to mix the mixture a little so the cells will not stay at the bottom of the vial) using the wide mouth of the syringe without needle (it is why round bottom of 2.2 ml Eppis is needed, the bottom of 1.5 ml Eppi is not wide enough to get the whole mix), then I switch to the wider blue needle (so the cells will not get destroyed due to the shear stress during injection). So, this way I fill one syringe per an animal, all the cells and Matrigel are taken in, I switch to the better needle and there is 100 ul of air behind the cell/Matrigel mix, so the mix does not touch the plunger. Then I put the syringe into ice in almost vertical position, load all the others and go to the animals.

I would use 50%matrigel/50% media. Make cells in the media containing half of total volume what you want. Add that to 50% matrigel. Place everything on ice, needles etc. Inject 100microlitre.

In my experience 20-50% matrigel works well depending on the cell line you want to inject. You can also inject 100-150 microliters. With subcutaneous injections when you inject its advisable to rotate your needle so that the bevel faces down before he pull out the needle. Injecting cells in matrigel is very easy, however, like most of the readers have suggested always keep everything cold. I would advice putting your needles on a dry ice bag all the time.

All procedural precautions have been already mentioned. My question it I have one querry that when we mix the cell line with matrigel and this cell suspension with matrigel can be injected s.c. How can we check thew uniformity of cells?

I am preparing the cell suspension by mixing 1 ml Matrigel with 1 ml of cell, from this I will take 0.2 ml for each mouse.Is this right procedure?

Durgesh, if you don't have too many mice to inject, i would recommend first aliquot your cell suspension required for each mice in an eppendorf (cold for obvious reasons) in PBS or plain medium and then add matrigel (1:1 or any other ratio depending on your experiment) or vice versa. This should take care of uniformity in your injections as you will be making cell suspensions for individual mice rather than taking a syringe full of matrigel and cell suspension and using it to inject in 5or 6 mice.

All the Best!

HI Durgesh

I do it pretty much as you explain - ie I make up enough for all my injections (and an extra 1-2 injections for pipetting error). For example, for 6 injections of 100 µL per injection, I make up enough cell suspension for 7 injections (ie 700 µL) to account for pipetting loss (which I find can be an issue with Matrigel).

I first make up 50% GFR Matrigel and 50% ice-cold PBS, mix well. Then I resuspend (without introducing bubbles) a cell pellet with this mix to give an appropriate cell dose per 100 µL and leave this on ice. Pretty soon after making the cell suspension (so as not to allow the cells to settle) I then use a 29G insulin syringe (with no dead space) to draw up and inject 100µL at a time into separate mouse flanks s.c.

I've tried both ways (individual preparations and using a mastermix) and I definitely find the mastermix approach much more reproducible - after all there's so much variability in mouse expts, the last thing you want to do is increase the technical error by making individual cell suspensions in my opinion.

Best of luck with your experiments

Al

Hi Alasdair,

Thanks for sharing your experience, but in my opinion, making aliquots of cell suspension (in PBS or medium) will still give better uniformity from injection to injection rather than resuspending cell pellet for 5 or 6 mice directly in a PBS: Matrigel mix, as chances of error in aliquoting correct amount of cell suspension (which is reasonably uniform, as we do routinely for MTS assay or other cell based assays in different 6well, 24 well or 96 well plate formats) are less as compared to injecting equal amounts of viscous matrigel mix in a 1 ml syringe.

But I agree with you, variability is part and parcel of such experiments and that’s why we take larger groups to remove these variabilities.

Regards

Pankaj

Hi Pankaj

I take your point regarding viscous solutions. But it is almost never more accurate to perform more pipetting steps when fewer will do (due to the compounding mechanical (and technichal) error). This is why we make mastermixes for PCR etc.

Specifically with the matrigel, the viscocity may well introduce increased error for accurate pipetting. However, if you keep the volumes large (100 µL) then it's easy to minimise this.

In the greater scheme of things I find a substantially greater source of variablity is the delivery method of the cell/matrigel suspension into the mouse. The amount of matrigel left in the syringe varies wildly depending on syringe and needle used. This error and the variablity in injection site. For this reason I use the insulin syringes noted above, and also increase my n values for each treatment to >6-8 (preferably 10).

bw

Al

Pls see Fridman et al., protocol in Nature, Vol. 7

there is a nice paper by R. Fridman et al in Nature protocols

Hi

I work constantly with matrigiel with a large variety of breast, colon, lung and melanoma cell lines. I have injected orthotopically and subcutaneously.

Use HC Matrigel at 50:50 with your cell suspension. KEEP EVERYTHING ON ICE and everything must aseptically handled.

Steps

1. calculate how much your total volume will be, for your mice number. Make an extra 1 ml for error etc. Matrigel is very viscous and you tend to lose some when pipetting and injecting.

2. Two days prior to use, place the matrigel in a box of ice and keep refrigrated at 4C, along with any pipette tips,  pipettes and vials, to be used.

3. on the day of injection, prepare your hood/work area, where injections are to be performed, and have the required number of syringes, on ice. All prior to preparing your cells.

4. Count/Prepare your cells into suspension.

5. Whatever your total volume required is, add cells and materigel at 50:50, for that total volume. Place cooled vials/eppendorfs, on ice and fill your eppendorfs (iml) with the mixed suspension. KEEP EVERYTHING ON ICE throughout your preparations.

6. Take your cells/matrigel eppendorfs, on ice, to your designated place of injection. If you are working in pairs, have one person preparing the syringes for 100ul ofr subcutaneous injection or 50ul for orthotopic injection, keeping the clean and filled syringes on ice, ready for the other person to inject the mouse. You have a 2 hour window from harvesting your cells to injecting the last animal.

~Hope this helps?

Hi,

I am trying to test the effect of an angiogenesis inhibitor in a matrigel in vivo plug. I am adding VEGF and heparin as control, and then adding several concentrations of my compound in which I expect to see a decrease in vessel formation.

should I use the growth-factor reduced matrigel, or the normal one? Thanks for your help

For tumor cells I would look at the Nature protocols paper by Fridamn a et al.  it has all the information, tips and uses.  Do not use growth factor reduced but use regular BME pathclear from trevigen or Matrigel from BD.

Hi.

Is there anybody ever try to use the stardard Matrigel (Cot. 354234) for in vivo cancerous cells injection please? We have this agent now and intend to try S.C. injection of MDAMB-231 to nude mice. The protein concentration would be around 9 mg/mL without PBS dilution that is similar to 1:1 diluted high protein Matrigel. But I'm afraid the standard Matrigel is too viscous to handle if it was used to mix cells pellet directly. 

Thanks for the advices

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?

Hi,

Does anyone know how long it takes for matrigel to degrade in NSG mouse after S.C. injection with cells (1:1)? And how long can matrigel hold the cells in place after S.C. injection (assuming the cells cannot form tumor by themselves)? Thanks!

Hi Shirley have you done a "dry run" or a test of your tumor cells in Matrigel in vitro to see how well they grow and if they form colonies?

If cells in the Matrigel blob surrounded by growth media do not grow and or form colonies, then it is doubtful that they will behave differently if injected SC.

Hi Steingrimur, thanks for the reply. Actually I am not using tumor cells in particular. I just want to know how long can the matrigel hold the cells in place after SC injection in mouse (assuming the cells cannot amplify by themselves), and how long does it take for the matrigel to degrade. I am not studying tumor growth or colony formation. Thanks.

Hi Shirley, I used to do a lot of IP Matrigel injections. 0.5-1 ml per mouse. If properly injected into the IP space, they would last a week or more as a big fat blob of Matrigel.

Sometimes I did not get a perfect IP injection. It was maybe half/half IP and SC.

In those mice, the Matrigel plug was tiny or not present at all after a week.

I hope this helps.

Matrigel must be stored cold. That's probably the most important aspect. Otherwise, adding the cells to the matrigel and injecting into Huh7 gives pretty stable results (for example: http://altogenlabs.com/xenograft-models/liver-cancer-xenograft/huh7-xenograft-model/). There are of course nuances with the injection, and the injection site needs to be chosen carefully for given research, but overall matrigel is meant to just be a buffer for the injection to help with tumor formation.

Instead of Matrigel whether Geltrex can be used for xenograft