Mouse endothelial cells were isolated from vehicle- and BGJ398-treated T241-Vector and T241–FGF-2 tumors to examine PDGF gene expression. Mouse pericytes were isolated from non-treated T241-Vector and -FGF-2 tumors to detect FGFR gene expression. Small pieces of tumor tissues were digested for 45 min at 37 °C with 0.15% collagenases 1 and 2 (Sigma-Aldrich; C0130, C6885). Single cells were stained on ice for 30 min with a rat anti-mouse CD31 antibody (553370; BD-Pharmingen) to bind to endothelial cells or a rabbit anti-mouse NG2 antibody (AB5320; Millipore) to bind to pericytes, followed by incubation for 15 min with a goat anti-rat Cy5 antibody (Millipore; AP183) or a goat anti-rabbit Cy5 antibody (AP132S; Millipore). Cells were washed with PBS and further incubated with anti-Cy5/Alexa Fluor 647 micro beads (130-091-395; Miltenyi Biotec) on ice for 10 min. Magnetic labeled cells were separated using magnetic columns and collected cells were stored in RNAlater (Sigma-Aldrich; R0901) at 4 or −20 °C until further use of gene expression study.
Ap183
Manufactured by Merck Group
Sourced in United States
The AP183 is a laboratory instrument designed for analytical testing and measurement. It serves as a versatile tool for researchers and scientists in various scientific fields. The core function of the AP183 is to provide accurate and reliable data collection and analysis capabilities to support scientific investigations.
Automatically generated - may contain errors
2 protocols using ap183
1
Isolation and Characterization of Tumor Cell Subpopulations
2
Isolation of Tumor Cell Subpopulations
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Tissue samples of PDGF‐B‐ or vector‐transfected LLC and T241 tumors were cut into small pieces and incubated with 1.5 mg/mL type I (17018029, Gibco) and 1.5 mg/mL II collagenase (17101015, Gibco) in PBS at 37°C for 40‐60 min. After filtering with a cell filter, cells were centrifugated at 1500 rpm for 10 min. Cell pellets were collected and incubated with a rabbit anti‐mouse NG2 antibody (1:50, AB5320, Millipore), a rat anti‐mouse CD31 antibody (1:50, 553370, BD Pharmingen, San Diego, CA, USA), a rat anti‐mouse PDGFRα antibody (1:50, 14‐1401‐82, eBioscience), a rat anti‐mouse PDGFRβ antibody (1:50, 14‐14012‐82, eBioscience), and a rat anti‐F4/80 antibody (1:200, NBP2‐12506, Novus Biologicals, Littleton, CO, USA), followed by species matched antibody incubation in ice, including a Cy5‐labeled goat anti‐rabbit antibody (1:200, AP132S; Millipore) or Cy5‐labeled goat anti‐rat antibody (1:200, AP183, Millipore) for 30 min. Cells were further washed and incubated with anti‐Cy5/Alexa Fluor 647‐coated microbeads (130‐091‐395, Miltenyi Biotec) for 15 min, and subsequently subjected to magnetic separation through magnetic columns. Collected positive cells were used for further study or stored in RNAlater (R0901, Sigma‐Aldrich, Burlington, MA, USA) at ‐70°C until further use.
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