Tumor organoids were isolated from primary tumors as described previously (5 (link)), with the ECM prepared as in (16 (link)). Stromal cells were isolated during the differential centrifugations that are used to separate organoids from the single cells. The supernatant from these centrifugation steps was collected and centrifuged at 1500 rpm for 5 minutes. The cell pellet was re-suspended in cell depletion buffer (PBS + 2% FCS + 1 mM EDTA), counted and diluted to 1×108 cells/ml. Biotin conjugated antibodies targeting CD326 (EpCAM; G8.8; 2 μg/ml; Biolegend), CD45 (30-F11; 2 μg/ml; Biolegend) and erythroid cells (TER-119; 1 μg/ml; STEMCELL Technologies) were used to deplete epithelial/immune/erythroid cells using the EasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit (STEMCELL Technologies), per the manufacturer’s instructions. The efficiency of cell depletion was assessed by flow cytometry using standard protocols. Details of this analysis are provided in Supplementary Materials and Methods .
Protocol detail
Isolation and Enrichment of Tumor Stromal Cells
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Antibodies
Biotin
Buffer
Centrifugation
Edta
Epcam
Epithelial cells
Erythroid cells
Flow cytometry
Isolation
Mouse
Organoids
Stemcell
Streptavidin
Stromal cells
Tumors
Corresponding Organization :
Other organizations : University of Southampton, Johns Hopkins Medicine, Johns Hopkins University
Top 5 similar protocols
Variable analysis
independent variables
- Preparation of extracellular matrix (ECM) as described in (16 (link))
- Efficiency of cell depletion assessed by flow cytometry
- Tumor organoids isolated from primary tumors as described previously (5 (link))
- Stromal cells isolated during the differential centrifugations used to separate organoids from single cells
- Cell depletion buffer (PBS + 2% FCS + 1 mM EDTA) used to re-suspend the cell pellet
- Biotin conjugated antibodies targeting CD326 (EpCAM), CD45, and erythroid cells (TER-119) used to deplete epithelial/immune/erythroid cells using the EasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!