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Preprint

Flow cytometry

YS Yi Sun
LC Lieping Chen
YZ Yuwen Zhu

Last updated date: Aug 17, 2021 Views: 769 Forks: 0

original An abbreviated version of this protocol was published in Science Translational Medicine
Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy
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Protocol for TIL isolation from fresh solid tumor tissue
• Wash tissue with 1XPBS to remove any blood.
• Chop tissue into small pieces as much as possible in RPMI complete media.
• Mechanically dissect tissue using the mouse- implant tumor C tube setting (gentile MACS dissociator).  (Optional)
• Add liberase DL (Roch) to medium at 50ug/ml and incubate for 40 min in 37°C on shaker.
• Repeat mechanical dissection. (Optional)
• Centrifuge, and filter out large particles through cell strainer (100um).
• Wash with complete media one time.
• Carefully load cell suspension onto the same volume of Ficoll in a 15ml (5ml each) or 50ml (20ml each) tube.
• Centrifuge at 1000xg for 30mins, at room temperature with brake-off.
• Collect cells in the middle layer, mix with >2X volume of medium before centrifuge down at 1000g for 5min.
• Wash with RPMI complete media once before proceeding for experiments.

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Sun, Y, Chen, L and Zhu, Y(2021). Flow cytometry. Bio-protocol Preprint. bio-protocol.org/prep1341.
  2. Sun, Y., Chen, W., Torphy, R. J., Yao, S., Zhu, G., Lin, R., Lugano, R., Miller, E. N., Fujiwara, Y., Bian, L., Zheng, L., Anand, S., Gao, F., Zhang, W., Ferrara, S. E., Goodspeed, A. E., Dimberg, A., Wang, X., Edil, B. H., Barnett, C. C., Schulick, R. D., Chen, L. and Zhu, Y.(2021). Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy. Science Translational Medicine 13(604). DOI: 10.1126/scitranslmed.abc8922

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