Fitc conjugated mouse anti human ck19 antibody

The expression of CK19 in breast cancer cell lines was determined by flow cytometry. In this regard, 1 × 10⁶ cells from each cell line were fixed with 4% paraformaldehyde (Merck) in PBS 1X for 20 min at room temperature. Then, cells were washed twice with 1% PBS with FBS and were permeabilized with ice-cold 100% methanol (Merck) for 30 min at 4°C. Cells were washed twice with 1% PBS-FBS and blocked with 1% bovine serum albumin (BSA) for 45 minutes at room temperature. Cells were washed twice with 1% PBS-FBS and then were stained with Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CK19 antibody (Abcam, diluted 1:300 in PBS 1X) or FITC-mouse IgG2a isotype antibody (Abcam, diluted 1:300 in PBS 1X) for 1h at room temperature as a negative control. Finally, they were washed twice with 1% PBS- FBS, and detection of bound antibodies was determined by flow cytometry (Cyflow). The results were analyzed with the FlowJo® program.

In this study, blood cells were fixed with 4% paraformaldehyde (Merck) in PBS 1× for 20 min at room temperature. Then, cells were washed twice with 1% PBS/FBS and were permeabilized with ice-cold 100% methanol (Merck) for 30 min at 4°C. Then, cells were washed twice with 1% PBS/FBS and were blocked and fixed with 1% Bovine Serum Albumin (BSA) for 45 min at room temperature. Then, cells were stained with the FITC-conjugated mouse anti-human CK19 antibody (Abcam, diluted 1:300 in PBS 1×) or FITC-mouse IgG2a isotype antibody (Abcam, diluted 1:300 in PBS 1×) as a negative control and incubated for 1 hr at room temperature. After twice washing with 1% PBS/FBS, detection of bound antibodies was determined by flow cytometry (Cyflow), and the results were analyzed with the following program.

CK19 expression in the MCF7 and T47D cell lines was also detected by immunofluorescence assay. Cultured cells were assessed on glass slides. They were fixed with 4% paraformaldehyde (Merck) in PBS 1X for 20 min at room temperature. The cells were washed twice with 1% PBS-FBS and were permeabilized with ice-cold 100% methanol (Merck) for 30 min at 4°C. Cells were washed twice with 1% PBS-FBS and blocked with 1% bovine serum albumin (BSA) for 45 min at room temperature. Cells were washed twice with 1% PBS-FBS, and then Immunostaining was done with the FITC-conjugated mouse anti-human CK19 antibody (Abcam, diluted 1:300 in PBS 1X). Then FITC-mouse IgG2a isotype antibody (Abcam, diluted 1:300 in PBS 1X) as a negative control was performed for 1h at room temperature. The cells were washed twice with 1% PBS-FBS, and detection of CK19 protein was determined under a fluorescent microscope (Leitz Laborlux).