Cd163 fitc

Cells were collected and incubated in PBS plus 0.1% BSA (9998S; Cell Signalling Technologies) and 5 µl Fc block per million cells for 20 min. 50 µl of cells were then incubated with 50 µl antibody cocktail diluted in PBS and 0.1% BSA for 20 min on ice in the dark. Cells were washed in 2 ml PBS and fixed in 2% PFA (15710; Electron Microscopy Sciences) diluted in PBS prior to analysis. Cells were analyzed on an LSRII flow cytometer. Antibodies were purchased from BD Biosciences Antibody (CD14-Alexa488, 562689; CD119-PE, 558934; CD86-BV421, 562433; CD11b-bv421, 562632; CD163-FITC, 563697; CD169-PE, 565248; CD206-APC, 561763; CD16-Alexa647, 557710; Alexa488 isotype, 557703; Alexa647 isotype, 57714; PE isotype, 12-4015-82; BV421 isotype, 562438; CD16-Alexa647, 557710; Alexa488 isotype, 557703). Flow cytometry data was analyzed and plotted in FlowJo (BD Biosciences).

Cells were collected and incubated in PBS plus 0.1% BSA (9998S; Cell Signalling Technologies) and 5 µl Fc block per million cells for 20 min. 50 µl of cells were then incubated with 50 µl antibody cocktail diluted in PBS and 0.1% BSA for 20 min on ice in the dark. Cells were washed in 2 ml PBS and fixed in 2% PFA (15710; Electron Microscopy Sciences) diluted in PBS prior to analysis. Cells were analyzed on an LSRII flow cytometer. Antibodies were purchased from BD Biosciences Antibody (CD14-Alexa488, 562689; CD119-PE, 558934; CD86-BV421, 562433; CD11b-bv421, 562632; CD163-FITC, 563697; CD169-PE, 565248; CD206-APC, 561763; CD16-Alexa647, 557710; Alexa488 isotype, 557703; Alexa647 isotype, 57714; PE isotype, 12-4015-82; BV421 isotype, 562438; CD16-Alexa647, 557710; Alexa488 isotype, 557703). Flow cytometry data was analyzed and plotted in FlowJo (BD Biosciences).

To collect cells, FACS buffer (DPBS + 1% BSA) was added to the Upcell Multi 24 well plate (method 4.5.1). The plates were kept at room temperature for 30 min to promote detachment of the cells and the cells were collected by pipetting the contents of each well. FcR blocking reagent was added to the cell suspension and the cell suspension was kept at room temperature for 15 min to inhibit Fc receptor-mediated nonspecific antibody binding. Each cell suspension was divided into two tubes. In one tube, the cells were mixed with the following six mouse monoclonal anti-human antibodies and the cell suspension was kept at 4 °C for 30 min for multiple staining: CD80-PE (Clone: L307.4), CD86-BUV395 (Clone: 2331), CD163-FITC (Clone: GHI/61), CD206-BV421 (Clone: 19.2), CD14-APC (Clone: M5E2), CD45-BV605 (Clone: HI30) (BD Bioscience). Cells in the other tube were mixed with nonspecific fluorescent mouse IgGs as a negative control. The cells were reacted at 4 °C for 30 min and then washed with FACS buffer. Data on the stained cells were acquired using a BD LSR Fortessa ™ Flow Cytometer (BD Bioscience) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).