Quantifying ANA Levels in Fcgr2b-Deficient Mice

The sera (1:400) of Fcgr2b-deficient mice were diluted at 8 months, and wild-type mice were tested for ANA as described^{11 (link)}. In short, diluted serum (30 µL) was added to the Hep-2 cell-coated slide, and phosphate-buffered saline (PBS) was used as a negative control, followed by incubation for 30 min. Then, the cells were washed with PBS 2 times and incubated with 30 µL of goat anti-mouse IgG–Alexa (1:500) (Abcam, Cambridge, MA, USA; cat. FA 1512-1010-1) for 30 min and washed with PBS. Finally, the slides were fixed and analyzed under a fluorescence microscope. The researcher will be blinded and grade the intensity as 4 = maximal fluorescence (brilliant yellow-green), 3 = less brilliant (yellow-green fluorescence), 2 = definite (dull yellow-green), and 1 = very dim (subdued fluorescence).