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CYTO_22158_sm_SuppFig1.tif24111KSupplemental Figure S1. Flow cytometry analysis of CD44-FITC titration. MDA-MB-468 tumor cell spiked blood samples (equivalent to 1% of the white blood cell count) were utilized to ensure appropriate tumor cell gating. MDA-MB-468 tumor cells spiked at a concentration equivalent to ∼0.01% white blood cells (∼200 MDA-MB-468 cells) were then incubated with various CD44-FITC volumes to MDA-MB-468 tumor cells incubated with 150μl of EGFR-FITC to determine the FITC mean fluorescence intensity, measured on a 4 decade log scale, signal-to-noise ratio that best matched that observed when analyzing 150μl of EGFR-FITC. Tumor cells were defined as CD44+CD45- or EGFR+CD45-.
CYTO_22158_sm_SuppFig2.tif21885KSupplemental Figure S2. Cell line comparison of EpCAM expression by FCM. (A) EpCAM expression of the MDA-MB-468, 21NT and LNCaP cell lines (n=3). (B) PE mean fluorescence intensity (MFI), measure on a 4 decade log scale, as an indicator of EpCAM antigen density, on the MDA-MB-468, 21NT and LNCaP cell lines (n=3). (C) FCM analysis of MDA-MB-468, 21NT and LNCaP cell lines incubated with anti-EpCAM-PE (red) and an IgG isotype control sample (blue).
CYTO_22158_sm_SuppFig3.tif21663KSupplemental Figure S3. Cell line comparison of CK8/18/19 expression by FCM. (A) CK8/18/19 expression of the MDA-MB-468, 21NT and LNCaP cell lines (n=3). (B) PE mean fluorescence intensity (MFI), measure on a 4 decade log scale, as an indicator of CK8/18/19 antigen density, on the MDA-MB-468, 21NT and LNCaP cell lines (n=3). (C) FCM analysis of MDA-MB-468, 21NT and LNCaP cell lines incubated with anti-CK8/18/19-PE (red) and a cells only control sample (blue).
CYTO_22158_sm_SuppFig4.tif3988KSupporting Information Figure 4. Supplemental Figure S4. Additional representative CellSearch® gallery images of optimized CD44-PE protocol from Figure 3. Representative CellSearch gallery images of 7.5ml of blood from a healthy volunteer donor, spiked with 1000 cells from the respective cell line, incubated with 1.5μg/ml of anti-CD44-PE, standard Veridex CXC reagents (CK-FITC, CD45-APC, DAPI), and analyzed at an exposure time of 0.2s. Orange squares indicate CD44+ CTCs, identified as CK+/DAPI+/CD45–/CD44-PE+.
CYTO_22158_sm_SuppFig5.tif3702KSupporting Information Figure 5. Supplemental Figure S5. Observed staining differences between CD44-FITC and M-30-FITC with the CellSearch® CTC kit. (A) Representative CellSearch® gallery images of 7.5ml of blood from a healthy volunteer donor, spiked with 4000 MDA-MB-468 human breast cancer cells treated with 0.1μg/ml of paclitaxel and subsequently incubated with 3.5μg/ml of anti-M-30-FITC. Orange squares indicate M-30+ CTCs, identified as CK+/DAPI+/CD45–/M-30+. (B) Representative CellSearch gallery images of 7.5ml of blood from a healthy volunteer donor, spiked with 1000 MDA-MB-468 human breast cancer cells, incubated with 4.0μg/ml of anti-CD44-FITC and standard Veridex CTC reagents (CK-PE, CD45-APC, DAPI), and analyzed with the CellSearch CTC kit and an exposure time of 0.5s. Orange squares indicate CD44+ CTCs, identified as CK+/DAPI+/CD45–/CD44-FITC+ (C) Representative CellSearch gallery images of 7.5ml of blood from a healthy volunteer donor, spiked with 1000 MDA-MB-468 human breast cancer cells, incubated with 1.5μg/ml of anti-CD44-PE and standard Veridex CXC reagents (CK-FITC, CD45-APC, DAPI), and analyzed with the CellSearch CXC kit and an exposure time of 0.2s. Orange squares indicate CD44+ CTCs, identified as CK+/DAPI+/CD45–/CD44-PE+.
MIFlowCyt_Checklist.doc51KSupporting Information: MIFlowCyt

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