Rat anti cd326 epcam apc cy7, by BioLegend - Product details

1

Cell Sorting and Antibody Staining

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Dissociated cells were stained with directly conjugated primary antibodies: rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend), goat anti-Trop2-APC (R &D Systems), rat anti-CD31-FITC (BioLegend), rat anti-CD45-FITC (BioLegend), and rat anti-Ter119-FITC (BioLegend) for 20 min on ice. Rat anti-ESAM-FITC (BioLegend) was also added to the Lin panel for some experiments. Rat anti-CD44-FITC (BioLegend) was used for analysis. Cells were stained in media containing RPM11640 (GIBCO), 10% FBS (Corning), 1x penicillin-streptomycin (GIBCO), and 10uM of the p160ROCK inhibitor Y-27632 dihydrochloride (Tocris Bioscience). Sorting was performed on a BD FACS Aria II (BD Biosciences) and flow cytometry analysis was performed on a BD FACS Canto (BD Biosciences).

Crowell P.D., Fox J.J., Hashimoto T., Diaz J.A., Navarro H.I., Henry G.H., Feldmar B.A., Lowe M.G., Garcia A.J., Wu Y.E., Sajed D.P., Strand D.W, & Goldstein A.S. (2019). Expansion of Luminal Progenitor Cells in the Aging Mouse and Human Prostate. Cell reports, 28(6), 1499-1510.e6.

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Multicolor Flow Cytometry of Mouse Prostate Cells

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Dissociated cells from mouse prostate were stained with rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend) and goat anti-Trop2-APC (R & D Systems) for 20 min on ice. Cells were washed with PBS and fixed in 1ml of 2% paraformaldehyde made from 16% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min on ice. Cells were washed with PBS and permeabilized in 1ml of permeabilization buffer (0.1% Saponin (Sigma-Aldrich), 5% FBS (Corning) in PBS) for 15 min at room temperature in the dark. Cells were resuspended in 100 μL of permeabilization buffer and stained with either rabbit anti-cytokeratin 5-Alexa Fluor 647 (Abcam) and rabbit anti-cytokeratin 8-Alexa Fluor 488 (Abcam), mouse anti-cytokeratin 14-FITC (Abcam), mouse anti-cytokeratin 18-FITC (Abcam) or rat anti-Ki67-FITC (BioLegend) for 20 min at room temperature in the dark. Cells were washed and resuspended in permeabilization buffer for analysis on a BD FACS Canto (BD Biosciences).

Crowell P.D., Fox J.J., Hashimoto T., Diaz J.A., Navarro H.I., Henry G.H., Feldmar B.A., Lowe M.G., Garcia A.J., Wu Y.E., Sajed D.P., Strand D.W, & Goldstein A.S. (2019). Expansion of Luminal Progenitor Cells in the Aging Mouse and Human Prostate. Cell reports, 28(6), 1499-1510.e6.

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Cell Sorting and Antibody Staining

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Dissociated cells were stained with directly conjugated primary antibodies: rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend), goat anti-Trop2-APC (R &D Systems), rat anti-CD31-FITC (BioLegend), rat anti-CD45-FITC (BioLegend), and rat anti-Ter119-FITC (BioLegend) for 20 min on ice. Rat anti-ESAM-FITC (BioLegend) was also added to the Lin panel for some experiments. Rat anti-CD44-FITC (BioLegend) was used for analysis. Cells were stained in media containing RPM11640 (GIBCO), 10% FBS (Corning), 1x penicillin-streptomycin (GIBCO), and 10uM of the p160ROCK inhibitor Y-27632 dihydrochloride (Tocris Bioscience). Sorting was performed on a BD FACS Aria II (BD Biosciences) and flow cytometry analysis was performed on a BD FACS Canto (BD Biosciences).

Crowell P.D., Fox J.J., Hashimoto T., Diaz J.A., Navarro H.I., Henry G.H., Feldmar B.A., Lowe M.G., Garcia A.J., Wu Y.E., Sajed D.P., Strand D.W, & Goldstein A.S. (2019). Expansion of Luminal Progenitor Cells in the Aging Mouse and Human Prostate. Cell reports, 28(6), 1499-1510.e6.

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4

Multicolor Flow Cytometry of Mouse Prostate Cells

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Dissociated cells from mouse prostate were stained with rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend) and goat anti-Trop2-APC (R & D Systems) for 20 min on ice. Cells were washed with PBS and fixed in 1ml of 2% paraformaldehyde made from 16% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min on ice. Cells were washed with PBS and permeabilized in 1ml of permeabilization buffer (0.1% Saponin (Sigma-Aldrich), 5% FBS (Corning) in PBS) for 15 min at room temperature in the dark. Cells were resuspended in 100 μL of permeabilization buffer and stained with either rabbit anti-cytokeratin 5-Alexa Fluor 647 (Abcam) and rabbit anti-cytokeratin 8-Alexa Fluor 488 (Abcam), mouse anti-cytokeratin 14-FITC (Abcam), mouse anti-cytokeratin 18-FITC (Abcam) or rat anti-Ki67-FITC (BioLegend) for 20 min at room temperature in the dark. Cells were washed and resuspended in permeabilization buffer for analysis on a BD FACS Canto (BD Biosciences).

Crowell P.D., Fox J.J., Hashimoto T., Diaz J.A., Navarro H.I., Henry G.H., Feldmar B.A., Lowe M.G., Garcia A.J., Wu Y.E., Sajed D.P., Strand D.W, & Goldstein A.S. (2019). Expansion of Luminal Progenitor Cells in the Aging Mouse and Human Prostate. Cell reports, 28(6), 1499-1510.e6.

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5

Prostate Cell Immunophenotyping by Flow

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Single-cell suspensions of 5 × 10⁴–1 × 10⁶ cells from mouse prostate tissue of different ages (each n = 4) were stained in cell staining buffer comprised of 1X DPBS (Gibco) with 5 g/L protease-free bovine serum albumin (Sigma-Aldrich) and 200 mg/L sodium azide (Sigma-Aldrich). Nonspecific antibody binding by Fc receptors was blocked using TruStain fcX (anti-mouse CD16/32) Antibody (BioLegend) according to the manufacturer’s protocol. Cells were then stained with rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend), goat anti-Trop2-APC (R&D Systems), and rat anti-CD45-FITC (BioLegend) for 30 minutes at room temperature. Cells were washed with cell staining buffer then fixed in 1% paraformaldehyde (Electron Microscopy Sciences) for 10–15 minutes at 37°C. After fixation, cells were chilled on ice for 1 minute, washed with cell staining buffer, and stored at 4°C before analysis on a FACSCanto flow cytometer (BD Biosciences).

Fox J.J., Hashimoto T., Navarro H.I., Garcia A.J., Shou B.L, & Goldstein A.S. (2023). Highly multiplexed immune profiling throughout adulthood reveals kinetics of lymphocyte infiltration in the aging mouse prostate. Aging (Albany NY), 15(9), 3356-3380.

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Rat anti cd326 epcam apc cy7

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5 protocols using rat anti cd326 epcam apc cy7

Cell Sorting and Antibody Staining

Multicolor Flow Cytometry of Mouse Prostate Cells

Cell Sorting and Antibody Staining

Multicolor Flow Cytometry of Mouse Prostate Cells

Prostate Cell Immunophenotyping by Flow

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