Kaluza flow cytometry data analysis software

Prior to staining, the cells were dissociated as described above. The cell suspension was carefully transferred (using a plastic Pasteur pipette) to 15 ml tubes. Due to the fragile nature of adipocytes, no washing steps were performed. One millilitre (1 ml) aliquots of the cell suspensions were stained with Nile Red (20 ng/ml final concentration), Vybrant® DyeCycle^TM Violet (VDC Violet; 2.5 µM final concentration) and mouse anti-human CD36-APC. After a 20 min incubation at room temperature, cells were analysed using a Gallios flow cytometer. Nile Red was excited by a 488 nm laser and fluorescent emission signals were collected using the FL2 (575/30 nm BP) detector. Non-induced ASCs were used to optimise the PMT voltage settings as well as to set the signal-to-noise threshold of the Nile Red fluorescence. After initial optimisation, all instrument settings were kept constant for the duration of the study. Flow Check^TM Pro (Beckman Coulter, Miami, USA) fluorospheres were run daily to validate instrument performance. Flow cytometry data was analysed using Kaluza flow cytometry data analysis software (Version 1.5, Beckman Coulter, Miami, USA).

Adipose-derived stromal cells (ASCs) were phenotyped at each passage. Prior to immunophenotype analysis, ASCs were stained with the following in Tube 1: CD34-PE Cy7, CD36-APC, CD45-Krome Orange, CD73-FITC and CD105-PE; and in Tube 2: CD44-APC Cy7, CD45-Krome Orange, CD73-FITC, CD90-PE Cy5 and CD105-PE. Cell viability was determined at each passage after staining with 7-AAD. Multi-parameter flow cytometry analyses were performed using a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA). Single color staining tubes were used to correct for spectral overlap of the fluorochromes (color compensation). Flow cytometry data was analysed using Kaluza flow cytometry data analysis software (Version 1.5, Beckman Coulter, Miami, USA).

Prior to 5 min of liver perfusion, latex beads (Invitrogen, FluoSpheres^®, 1.0 µm diameter, carboxylate-modified) at 0.57 µl/g body weight were injected in the tail vein. Following liver perfusion, the cells were isolated. KC surface marker F4/80 was stained. Percentage of phagocytic cells and fluorescent intensity of latex beads in F4/80-positive cells were evaluated by flow cytometry.
The expression levels of KC surface proteins involved in phagocytosis (CD68, macrophage receptor with collagenous structure; MARCO, macrophage class A scavenger receptors; SR-A) were analysed by flow cytometry. Samples were incubated with APC-conjugated anti-F4/80 (17-4801-82, eBioscience), PerCP/Cy5.5- conjugated anti-CD68 (137010, BioLegend), MARCO (MCA1849, Bio-Rad) and SR-A (AF1797, R&D Systems), followed by incubation with the secondary antibody Alexa Fluor 488 (Invitrogen). All flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter). Data were analysed using Kaluza flow cytometry data analysis software (v. 1.2, Beckman Coulter).