Quantifying Nuclear A3B Expression

1.0 × 10⁶ THP18 (WT), THP18-ΔA3A, and THP18-ΔA3A/B cells were seeded in 6-well plates, allowed to grow for 24 h, and were then induced with 1 µg/mL lipopolysaccharide (LPS) (MilliporeSigma, #L2654) and 300 U/mL of Interferon-alpha (IFN-α) (R&D Systems Inc., Minneapolis, MN, USA, #11200-1) for 24 h. The 293T and inducible T-REx-293-A3B-eGFP cells were also included in the CMA, with the latter induced for 24 h with 1µg/µL doxycycline. Cell buttons were prepared as follows: Trypsinization, centrifugation, and supernatant removals were followed by 3 washes with PBS. Following the last wash, cells were resuspended in 10% formalin for 15 min. After fixation, cells were rinsed 3× with PBS, mixed with Histogel (Thermo Fisher Scientific, #HG-4000-012) at 65 °C, and allowed to solidify at RT for approximately 10 min. The resulting cell blocks were placed in Histosette II cassettes, dehydrated, and embedded in paraffin.
Immunohistochemical staining of the CMA was performed as above for tissues. Nuclear A3B immunoreactivity was visualized with the Aperio ScanScope XT (Leica Biosystems) and quantified using the Aperio Nuclear Algorithm. Histoscore (H-Score) was calculated using the formula (3+) × 3 + (2+) × 2 + (1+) × 1, as described earlier [56 (link),57 (link)].