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ES细胞培养-实验方法

作者:高考题库网
来源:https://www.bjmy2z.cn/gaokao
2021-02-16 09:01
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-

2021年2月16日发(作者:荷马史诗英文)


ES


细胞培养


-


实验方 法




ES cell culture media and solutions



ES


细胞培养需要较高的实验条件,培养血清要用纯度较高的(


ES


级别)的胎牛血清,为防


止细胞分化,需要在培养皿底部 铺种滋养层细胞(


Feeder cells


)并在培养基中加 入白细


胞抑制因子(


LIF



。培养细胞的平皿和吸管均为一次性聚乙烯材料。



(A)



D


MEM with high glucose


(B)



0


.1


mM


non-essential


amino


acids


(100


×


stock,aliquoted


,stored


at


4



)


(C)



1


mM sodium pyruvate (100


×


stock,aliquoted ,stored at 4



)


(D)



1


0


-4


M


β


?


mercaptoethanol (100


×


stock,aliquoted ,stored at-20



)


(E)



2


mM L


?


glutamine (100


×


stock,aliquoted ,stored at-20



)


(F)



1


5%FBS


要用纯度较高的(


ES


级别)的胎牛血清



(G)



P


enicillin and streptomycin (final concentration 50


μ


g/ml each)


(H)



1


000 U/ml LIF


白细胞抑制因子,抑制


ES


细胞分化





Preparation of EMFI feeder layers


Regents


Frozen vials of primary embryo fibroblasts


Tissue culture dishes


PBS without Ca2+ and Mg 2+


0.05% tripsin in saline /EDTA


DMEM +10%FBS


Mitomycin C (stock 1 mg/ml in PBS stored in dark at 4



and used within


two


weeks


,mitomycin


C


is


toxic


;wear


gloves


and


use


caution


when


handling )



Methods


1.



Thaw a frozen vial EMFI cells quickly at 37



.


2.



Add cells to 10 ml DMEM +10%FBS and centrifuge (270


g,


5 min)



3.



Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM


+10%FBS, and


split


onto five


150


mm plates each containing a total of


25


ml


DMEM


+10%FBS


.Mix


well.


注意摇动混匀,不要单纯只按一个方向摇动,


以使细胞较均匀地分布于平皿中,




4.



Incubate cells at 37



,5% CO


2 .



5.



When the cells form a confluent monolayer (approx. three days )each


plate should either be:



(a)Thrypsinized


,split


onto


five


additional


150


mm


dishes


,and


grown


until they form a confluent monolayer ,or


(b)Directly treated with mitomycin C


(丝裂霉素)


to inhibit cell


growth and division .


因为

< p>
ES


细胞与滋养细胞共培养时,未经丝裂霉


素处理 的滋养细胞增殖很快,


会与


ES


细胞竞 争养分,


此步丝裂霉素处理


可使滋养细胞失去增殖,但仍保持存 活。



6.



Remove the medium from the confluent plates and 10 ml DMEM + 10%FBS


containing 100


μ


l mitomycin C (1mg/ml stock).Swirl plates to ensure


an even distribution of medium.


7.



Incubate cells at 37



,5% CO


2


for 2-2.5h.


8.



Wash the monolayer of cells twice with 10 ml PBS per dish .


9.



Add 5 ml trypsin /EDTA to each plate .


10.



incubate 37



,5% CO


2


until the cells come off the plate .


11.



Add 10 ml DMEM +10%FBS to each plate and break any cell aggregates


by


gently pipetting.


12.



Centrifuge


cells


(270


g,5min)and


resuspend


the


pellet


in


DMEM+10%FBS.


5


13.



Count the cells and dilute to a concentration of 2


×


10


cells/ml.


14.



Plate the cells immediately onto tissue culture dishes containing


DMEM+10%FBS for the appropriate cell densities and volumes of medium


for different plate sizes.


15.



Allow


feeders


to


attach


at


least


2h


,but


preferably


overnight


,before


adding ES cells.


16.



Change the medium to ES cell medium immediately before adding ES


cells .Mitomycin C treated EMFI feeders can be used for up to seven


days with medium changes every three to four days.






Preparatiooon of a stock of mitomycin C treated EMFI cells


Reagents


正常的滋养细胞贴壁生长于培养皿底面,呈 梭形,应当均匀分布,并将皿底完


全覆盖。



1.



thaw a frozen via of EMFI cells quickly at 37



.


2.



Add cells to 10 ml DMEM +10% FBS and centrifuge (270 g,5min).


3.



Decant


supernatant


,


resuspend


the


cell


pellet


gently


in


10


ml


DMEM


+10%FBS, and split onto five 150 mm plates each containing a total


of 25 ml DMEM +10%FBS .Mix well.



4.



incubate cells at 37



,5% CO


2 .



5.



When


the


cells


form


a


confluent


monolayer


(approx.


three


days


)each


plate


should


trypsinized


,split


onto


five


additional


150


mm


dishes ,and grown until they form a confluent monolayer


6.



Remove the


medium


from the


confluent


plates and 10


ml DMEM +


10%FBS


containing


100


μ


l


mitomycin


C


(1mg/ml


stock).Swirl


plates


to


ensure


an even distribution of medium.


7.



Incubate cells at 37



,5% CO


2


for 2-2.5h.


8.



Wash the monolayer of cells twice with 10 ml PBS per dish .


9.



Add 5 ml trypsin /EDTA to each plate .


te 37



,5% CO


2


until the cells come off the plate .(5-10min).


10 ml DMEM +10%FBS to each plate and break any cell aggregates


by gently pipetting.


fuge cells (270 g,5min)and resuspend the pellet in freezing


medium


.Freezing


all


the


cells


from


each


plate


in


one


freezing


vial


in


1


ml


of


1


×


freezing


medium


and


store


at


-70



for


one


day


.Transfer


the vials to liquid nitrogen.



make


feeder


plates


thaw


a


frozen


vial


of


mitomycin


C


treated


EMFI


cells quickly at 37




cells to 10 ml DMEM +10%FBS and cenrifuge (270 g,5min).


supernatant , and resuspend the cell pellet gently in 30 ml


DMEM +10%FBS.



cells


directly


into


tissue


culture


plates.


Depending


of


the


size


of


the


plates


required


put


10


ml


/100mm


plate,


5ml


/60


mm


plate,5ml/60


mm plate, or 1.5ml/35mm plate.



feeders


to


attach


preferably


overnight


,before


adding


ES


cells


or at least 2h if using gelatinized plates .


the medium to ES cell medium immediately before adding ES


cells .




Growth of ES cells on feeders plates


Equipment and reagents



100mm dishes containing feeder layers


ES cell medium briefly pre-warmed to 37




0.



05% trypsin in saline /EDTA


PBS without Ca2+ and Mg2+


Gelatin ,0.1% solution in water ,autoclaved



Method


1.



Quickly


thaw


one


vial


of


frozen


ES


by


warming


in


your


hand


or


at


37



,and


transfer


cells


to


a


12


ml


tube


containing


10


ml


of


ES


cell


medium


before


all the ice has disappeared .


2.



Centrifuge at 270 g for 5min .


3.



Aspirate


supernatant


and


resuspend


pellet


in


10


ml


ES


cell


medium


and


plate on a 100 mm dish with a feeder layer.


4.



Change


the


medium


the


next


day


by


swirling


the


medium


in


dish


to


collect


debris


,then


aspirate


.Add


fresh


medium


gently


to


the


side


of


the


plate


so that the feeder layer is not disturbed.


5.



On


the


second


day


(cell


should


be


just


subconfluent)


wash


cells


twice


with PBS and add 2 ml trypsin/EDTA.


6.



Incubate for approx .5 min at 37



until cells begin to come off the


plate .

-


-


-


-


-


-


-


-



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