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楼主 |
发表于 2017-11-13 20:27:21
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1. Digestion solution by Miltenyi degestion machine（no need add DNase）
a. Prepare Enzyme D by reconstitution of the lyophilized powder in each vial with 3 mL of RPMI 1640 or DMEM. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution. For cell culture experiments subsequent to tissue dissociation, Enzyme D should be sterile filtered prior to aliquoted.
b. Prepare Enzyme R by reconstitution of the lyophilized powder in the vial with 2.7 mL RPMI 1640 or DMEM. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.
▲ Note: Make sure to thoroughly mix this suspension immediately before withdrawing the required reaction volume!
c. Prepare Enzyme A by reconstitution of the lyophilized powder in the vial with 1 mL of Buffer A supplied with the kit. Do not vortex. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.
d. Prepare enzyme mix by adding 2.35 mL of RPMI 1640 or DMEM, 100 μL of Enzyme D, 50 μL of Enzyme R, and 12.5 μL of Enzyme A into a GentleMACS C-Tube.
e. <0.6g tumor for 2.35 mL Digestion solution each C-Tube.
2. Digestion solution from Invitrogen
a. Weight Collagenase I，II，IV each 5mg,
b. pour them into 10ml RPMI1640, Add 100ul of 100x DNase (20mg/ml),mix well，
c. <0.6g tumor for 5 mL Digestion solution
3. Digestion solution from Roche
a. Re-suspend 5mg of Liberase (26 Wunsch Units/vial) in 15ml of RPMI1640.
b. Re-suspend for <30min at 4℃ with gentle agitation. Add 150ul of 100x DNase (20mg/ml).
以上是我们比较常用的方法，但是消化肿瘤的活率跟很多因素有关系。比如和你得到的肿瘤的质量，大小的关系都非常大。 |
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发表于 2017-11-12 11:22:41
发表于 2017-11-13 10:02:32
