The CXCR4 expression profiles in the two types of BMSCs at two passages (P3 and P6) were assessed by immunofluorescence staining. Cells were collected and fixed in 4% paraformaldehyde at 4°C for 20 min. Following treatment with 0.5% Triton X-100 and 1% bovine serum albumin (Thermo Fisher Scientific, Inc.) for 1 h at 25°C, cells were incubated with rabbit anti-CXCR4 antibody (ab124824; 1:100; Abcam, Cambridge, MA, USA) overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor® 488-conjugated anti-CXCR4 antibody (ab208128; Abcam) for 30 min at 37°C followed by DAPI (Sigma-Aldrich; Merck KGaA) staining for 2 min, to counterstain the cell nuclei. The sections were mounted and then observed under a Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) and the intensity of the CXCR4-positive area were measured using Image-Pro Plus 6.0 software. The surface expression of CXCR4 was also analyzed using flow cytometric analysis as previously described (22 (link)). The cells were incubated with FITC-conjugated anti-CXCR4 antibody (Abcam).
Ab208128
Manufactured by Abcam
Ab208128 is an antibody that can be used for various laboratory applications. It is a polyclonal antibody raised against a specific target antigen. The core function of this product is to detect and bind to the target antigen in a sample, allowing for its identification and quantification.
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2 protocols using ab208128
1
Profiling CXCR4 Expression in BMSC Subsets
2
Detecting CXCR4 in Pancreatic NET Cells
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In order to develop the assay for CXCR4 detection using the CellSearch platform, the pancreatic NET (pNET) cell line BON1 was used. This has previously been reported to express both EpCAM and CXCR4.12 (link) Five hundred BON1 cells were spiked into 7.5 mL of healthy donor blood collected in CellSave preservative tubes and processed through the CellSearch platform. A fluorescein-conjugated antibody can be added for detection by the fourth fluorescence channel to further characterise the cells for an additional marker of interest. For this study, the AF488-conjugated anti-CXCR4 antibody [UMB2, Abcam, Ab208128] was used and cells were defined as positive for CXCR4 expression when staining was present in the fourth channel (Fig. 1a, b ). Patient samples were analysed using the protocol optimised on cells (antibody concentration 1:10 and exposure time 5 s), and evaluations regarding the expression of CXCR4 on CTCs were made by two independent operators having no knowledge of the clinical status of the patients.![]()
CellTracks Analyzer II example images.
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