Goat anti rabbit igg h l cy5

The cell suspension obtained from P1-MEF/KSF-HFC cells following digestion was filtered through a 40-µm filter to remove impurities, followed by centrifugation at 500 g or 5 min. Flow cytometry staining was subsequently performed (on the ice). Cell pellets were washed, resuspended in ice-cold fluorescence-activated cell sorting (FACS) buffer (pH 7.4; 0.1 M PBS; 1 mM EDTA and 1% BSA) and stained for markers with the following antibodies: Pan-cytokeratin (CK; 1:1,000; anti-pan Cytokeratin antibody; cat. no. ab234297; Abcam), α-SMA (1:1,000; cat. no. ab5694; Abcam) and NCAM (1:1,000; cat. no. ab204446; Abcam) were added for 30 min at 4˚C. PBS buffer was used for the control group. The cells were washed twice with PBS and centrifuged at 500 x g for 5 min at 4˚C. Subsequently, goat anti-rabbit (1:1,000) antibodies including: (Alexa Fluor^® 488; cat. no. ab150077; Abcam), Goat Anti-Rabbit IgG H&L (Cy5^®; cat. no. ab6564; Abcam) and Goat Anti-Rabbit IgG (FITC; cat. no. ab6717) were added and incubated for 30 min in the dark at 4˚C. Samples were rinsed twice with PBS, centrifuged at 500 x g or 5 min at 4˚C, resuspended in PBS containing 2% FBS and prepared for further analysis. The cells were analyzed using flow cytometry analysis software Flow Jo7.6.5 (FlowJo LLC).