MatriGel plating - (Mar/21/2007 )
Hello,
I am working with a colleague and we are attempting to spread dilute MatriGel onto 12 well plates. We are trying to obtain a thin, even coating on the bottom of the well. The problem is getting an even spread to coat the entire well. Is there a way to "spread" MatriGel across the plate? There seem to be some areas where it simply doesn't want to coat, while it hangs out on the side. So, there is a sufficient amount of MatriGel it just won't behave.
Thanks
Amelia
You simply have to shake the plate gently to cover all regions in the plate.
we add matrigel and once the whole surface is covered, we remove matrigel and leave it to dry. thats how we coat plates with matrigel.
how much of matrigel do you use for each well.
we add matrigel and once the whole surface is covered, we remove matrigel and leave it to dry. thats how we coat plates with matrigel.
how much of matrigel do you use for each well.
We were using 75ul for each well of a 12 well plate, but it never wants to coat the entire surface, this includes tilting the plate gently. Do you do this with your plate on ice?
we add matrigel and once the whole surface is covered, we remove matrigel and leave it to dry. thats how we coat plates with matrigel.
how much of matrigel do you use for each well.
We were using 75ul for each well of a 12 well plate, but it never wants to coat the entire surface, this includes tilting the plate gently. Do you do this with your plate on ice?
No we dont do it on ice.
we use 150ul/ well in 24 well plate. we keep tilting the plate after we add matrigel and also while we remove it.
we add matrigel and once the whole surface is covered, we remove matrigel and leave it to dry. thats how we coat plates with matrigel.
how much of matrigel do you use for each well.
We were using 75ul for each well of a 12 well plate, but it never wants to coat the entire surface, this includes tilting the plate gently. Do you do this with your plate on ice?
No we dont do it on ice.
we use 150ul/ well in 24 well plate. we keep tilting the plate after we add matrigel and also while we remove it.
Okie dokie. Thanks. I think we just need practice then
Amelia
there is no need for practice here, its quite simple.
I think you prepare matrigel in a different way than we do here, so the difference.
good luck
I am working with a colleague and we are attempting to spread dilute MatriGel onto 12 well plates. We are trying to obtain a thin, even coating on the bottom of the well. The problem is getting an even spread to coat the entire well. Is there a way to "spread" MatriGel across the plate? There seem to be some areas where it simply doesn't want to coat, while it hangs out on the side. So, there is a sufficient amount of MatriGel it just won't behave.
Thanks
Amelia
Matrigel must be well shaked, and re-shaked after it has been thawn; do not take more than 20 µl per well of a 12 well plate; as Matrigel is viscous, spread it with a pipet tip or a policeman
To coat plates with Matrigel, we incline the plate and we place the head of the tip on the higher board of the well. Then we release gently the Matrigel, it runs on the well surface and cover it. If needed we shake gently the plate for a complete coverage (but it's not always necessary). We use a mininum of 330µl undiluted Matrigel for a well of a 24-well plate.
To work on ice with the plate is better but not necessary if you work quickly (Matrigel bottle has to be kept on ice!). We refrigerate all the material that will be in direct contact with the Matrigel (plates, tips,...) previous to use.
Hope this may help.
I am working with a colleague and we are attempting to spread dilute MatriGel onto 12 well plates. We are trying to obtain a thin, even coating on the bottom of the well. The problem is getting an even spread to coat the entire well. Is there a way to "spread" MatriGel across the plate? There seem to be some areas where it simply doesn't want to coat, while it hangs out on the side. So, there is a sufficient amount of MatriGel it just won't behave.
Thanks
Amelia
Well well! Here some additions to Matrigel coating from my experience
I've studied invasion of tumor cells and coated BD cell culture inserts ( for 24 well plates). I 've used BD MAtrigel(Matrigel GFR, Becton Dickinson). Stock solution contained 10mg\ml Matrigel in 5 ml. I recommend you to do as The Bearer said " Matrigel must be well shaked, and re-shaked after it has been thawn; as Matrigel is viscous, spread it with a pipet tip or a policeman". If you are a beginner in working with Matrigel make all operation on ice. The product is expensive and so you should not risk on your first steps. When you plan your exp try to estimate what quantity of M you need for one exp, aliqote on ice and keep frozen.
For my purpose I've diluted stock solution ( to 1mg\ml with dilution buffer ( previously cool till 4 C ). To coat cell culture insert ( area near 1\2 of well of 24 plate ) my volume was 40 ul. I've dropped "tear" in the center of insert then quickly with tip spread this volume to coat all surface.
About dilution - it depends on your purpose.
About dilution buffer: I've used the following buffer: Tris ( 40mM) sucrose ( 4%) NaCl ( 30mM ) adjust to pH 8.0)
I will try to explain the composition of this buffer:
For uniformly polymerization of matrigel it is necessary to provide optimal rate of aggregation of connective tissue proteins to avoid formation of protein aggregates on surface. It is well known that this process is controlled by pH. Optimal pH in alkaline range and near 8.0. So Tris buffer is a choice. Nacl - is additive also to prevent spontaneous aggregation. Sucrose was added to regular or uniform salt cristalization during drying polymer layer.
When you coat surface - let dry at 37 C for 2 hour ( 40-60% humidity, best in CO2 incubator) then complete drying at 30 C and same humidity for 24 hours. Then you can store you plate at 4 C. Before you add your cells you should rehydrate polymer layer for 2 Hours in CO2 incub., adding culture medium.
To check quality of your coating use Coomassie Staining (0.25g Coomassie R-250, 10ml MeOH, 30ml Acetic acid). Incubate 5 minutes, wash and check under microscope. Make it with one of you control well. Some people then make destaining and use wells for exp. But I think it is not very good.
The behavior of cells on Matrigel sometimes spellbind
Good luck!
If you dilute the matrigel in DMEM (1:40) and use it, you dont need to work on ice and matrigel is more manageable.
Also i dont think you lose any of the matrigel properties as we get decent cultures on matrigel.