Single cell suspension was obtained as previously described (34 (link)). The cells were stained with antibodies, purchased from either BioLegend [anti-CD3e (145-2C11, RRID: AB_312660), anti-CD8𝛼 (53-6.7, RRID: AB_2888883), anti-CD11b (M1/70, RRID: AB_312791), anti-CD25 (PC61.5, RRID: AB_312847), anti-CD29 (HMβ1-1, RRID: AB_312885), anti-CD31 (390, RRID: AB_10613644), anti-CD45 (30-F11, RRID: AB_2563598), anti-CD140a (PDGFRα, RRID: AB_1953268, APA5), anti-CD324 (EpCAM, G8.8, RRID: AB_1134172), anti-VEGFR3 (AFL4, AB_10680790), and anti-PDPN (8.1.1, RRID: AB_2629802)] or from Thermo Fisher Scientific [anti-CD4 (RM4-5, RRID: AB_464902), anti-CD69 (H1.2F3, RRID: AB_1210795), anti-FoxP3 (FJK-16s, RRID:AB_467575), and anti-NK1.1 (PK136, RRID: AB_2536075)] or from Abcam [anti-αSMA (EPR5368, RRID: AB_11129103)]. Cells were also stained with Fixable Viability Dye (Thermo Fisher Scientific) and dead cells were gated out from the analysis. Cell types were determined as following; lymphatic cells: CD45+/CD3+, CD8 T+ cells: CD45+/CD3+/CD8+, Tregs: CD45+/CD3+/CD4+/FoxP3+, cancer cells (MOC1): CD45−/ CD31−/EpCAM+/CD140a−/VEGFR3−, cancer cells (MC38-luc): CD45−/CD31−/CD29+/αSMA−, CAFs (MOC1): CD45−/CD31−/EpCAM−/CD140a+/VEGFR3−, CAFs (MC38-luc): CD45−/CD31−/αSMA+, vascular endothelial cells: CD45−/CD31+/EpCAM−/CD140a−/VEGFR3−, lymphatic endothelial cells: CD45−/CD31−/EpCAM−/CD140a−/VEGFR3+.
Protocol detail
Multiparametric Flow Cytometry Analysis
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.
Corresponding Organization : Center for Cancer Research
Top 5 similar protocols
Variable analysis
independent variables
- Antibodies used for cell staining
- Cell types determined based on marker expression
- Proportions of different cell types (lymphatic cells, CD8 T+ cells, Tregs, cancer cells, CAFs, vascular endothelial cells, lymphatic endothelial cells)
- Cell suspension preparation (previously described in reference 34)
- Fixable Viability Dye for exclusion of dead cells
- Positive control: None specified
- Negative control: None specified
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!