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1.Remove a 10 ml bottle of Matrigel from the -80 freezer, and place it directly on

wet ice. Cover the bottle with ice, in ice bucket, and put it in the fridge to thaw.


2.Thaw overnight (recommended), or a few hours until entirely Matrigel contents

are liquid.


3.Do not allow the bottle to be exposed to ambient air above 14 degrees C, the

gelling temperature of matrigel. While handling during this procedure, the bottle must be constantly immersed in ice.


4.Look up the specification sheet for this particular Matrigel lot in the Growth

factor binder. The specification sheet will list the protein concentration in mg/ml.

Matrigel is a crude mixture of protein, see BD’s Matrigel spec sheet for details.

Typically, protein concentration will vary between lots, so make sure to check

specs for the correct lot that you are using.



5.Each tube needs to be aliquotted so as to be the correct amount to coat 6 well of 6-

well plate, so, each tube of Matrigel needs to contain 2 mg protein. Therefore, a

calculation should be made to determine the volume to add to each tube.

2mg/(conc. in mg/ml) = A ml (the mgs cancel), where A = aliquot volume.

Example calculation: example Matrigel bottle comes at 14.5 mg/ml.

we want 2 mg/tube. 2 mg/14.5mg/ml=0.138ml. Then add 0.138 ml / tube.


2mg/(conc. in mg/ml) = A ml (the mgs cancel), A = 加入液体量.

计算举例:每瓶购入的Matrigel的蛋白量为14.5mg/ml,分装后的为2mg/管,2 mg/14.5mg/ml=0.138ml.每管加入的Matrigel量为0.138ml。

6.When bottle is thawed, remove the ice bucket with bottle and place them in the

TC hood.


7.Additionally, prepare a shallow bucket filled about 2/3 full and patted down with

wet ice and put it in the TC hood.



http://www.wendangku.net/doc/1f3bf8e2172ded630a1cb603.htmling sterile microfuge tubes, open tubes sterile and stick each of them in the

shallow bucket ice bath. Arranging the tubes into rows is easiest to handle.


9.Open the Matrigel bottle with pliers and sterile pipet aliquots with P1000

pipetman. Change tips after every few aliquots. Tips may be chilled in advance to reduce gelling in tip during aliquotting, though if aliquotting is done fast this is

not necessary.



10.Cap each microfuge tube and place in a carboard box in the -80 degree, labeled

with aliquot size, lot number, date and name.

盖上离心管盖,置-80℃预冷的储存盒内(carboard box),标记好体积,批号,日期和名称。

11.Transfer tubes from wet ice to -80 ultra-low freezer without exposure to room

temperature air as the tubes can warm up and gel very fast.


Plate Matrigel


1.Remove one tube of Matrigel from the -80 per 6-well plate of matrigel needed.

Do not allow matrigel to be exposed to ambient temperature.


2.Place the tube in the fridge for at least one hour to thaw.


3.Remove tube from fridge, keep the tube on ice or in a bench chiller. Take to

biosafty cabinet.


4.Take out a 15 ml conical tube. Using a 5 ml glass pipet, add 6 mls of cold DF12

medium to the 15 ml tube. This will be used to dilute the Matrigel aliquot.


5.Add the contents of the Matrigel tube to the medium. This can be done with the

same 5 ml pipet. A P-1000 pipetman or a 2 ml glass pipet may also be used for this step.

将Matrigel加入到培养基中。该操作可以使用同一根5ml吸管,也可以使用P-1000 自动移液器或 2 ml 玻璃吸管。

6.Mix the Matrigel with the DF12 by pipetting about 3 times with a 5 ml pipet. Try

not to cause bubbles.


7.Suck up to 6 mls and dispense one well to each well of a 6-well plate. If desired,

separate wells to different plates, for example, 3 well on each of two plates.


8.Agitate plate so that Matrigel solution covers the entire well and dose not miss

patches of well surface.


9.Leave plate to gel for at least one hour at room temperature.


10.If plate is not to be used, it may be wrapped in plastic wrap or parafilm, and

stored in the fridge for up to one week.


11.When about to split cells on to Matrigel plate, aspirate excess Matrigel right