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翻译Matrigel

Aliquote Matrigel

分装Matrigel

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.

从-80冰箱中取出装有10ml的Matrigel瓶,直接置于冰上。(在冰桶中装满冰,Matrigel置于其上,然后放置在冰箱中解冻。)

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

are liquid.

过夜解冻(推荐),或数小时解冻后直至Matrigel完全解冻成液体状态。

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.

不要将Matrigel暴露在14℃以上的空气环境中(成胶温度),在整个操作过程中,Matrigel瓶必需始终置于冰桶中。

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.

阅读本批Matrigel的批次说明书,说明书中列出了蛋白的浓度(mg/ml)。

Matrigel是未经加工的原始的蛋白混合物,具体见BD公司说明书(Matrigel为动物基质瘤细胞的分泌蛋白产物)。一般来说,不同批次的Matrigel其蛋白含量是有差异的,因此一定在使用前仔细查看本批次的蛋白含量。

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.

Matrigel可以按能正确包被一个6孔板的量来分装至小管中。因此,每管应该含有2mg的蛋白量,所以需要通过计算来确定每管的分装液体量。

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.

当Matrigel解冻后,将冰桶和Matrigel一起移到超净台内。

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

wet ice and put it in the TC hood.

另外,准备一个浅的冰盒,装上2/3量的冰,然后置于超净台内(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.

将无菌的微离心管打开,成排插入到浅冰桶中,以方便取用。

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.

用钳子打开Matrigel瓶盖,用P1000自动移液器分装,分装数管后应更换用新的Tip。Tip事前需预冷以减少在分装过程中成胶作用。不过,如果分装

很快,Tip预冷也不是一定必需的。

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.

将离心管从冰中转移到-80℃超低温冰箱中,由于离心管升温和成胶很快,避免暴露在室温空气中。

Plate Matrigel

铺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.

从-80℃取出铺6空板需要的一管Matrigel置冰上,不要将Matrigel暴露在室温下。

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

将离心管置冰箱中解冻至少1小时。

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

biosafty cabinet.

从冰箱中取出离心管,将离心管置于冰上或冷却器中,然后置于生物安全橱。

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.

取1个15ml锥形管,用5ml玻璃吸管加入6ml冷DF12培养基入15ml离心管中。

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.

用5ml玻璃吸管吹打3次左右,混匀Matrigel和DF12,不要吹打出气泡。

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.

吸取6ml混合好的液体加入到6空板内的每个孔。如果有其他需求,也可以加入到不同6空板内的不同孔中,2快板,每个板3个孔。

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

patches of well surface.

晃动培养板,使得Matrigel覆盖整个孔,不要有空留部分。

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

室温下至少包被处理1小时。

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.

如果培养板不使用,可以将之用塑料膜或石蜡膜包裹,在冰箱中可以保存一个星期(Matrigel不用吸出)。

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

beforehand.

当将细胞传代到Matrigel包被的培养板时,只需在使用前吸除添加的

Matrigel即可。