Figure 7.

No IgG- or IgE-receptor expression was found in P-STS cells (age of culture < 6 months). (A) RBL SX-38 cells or Il-4 treated P-STS cells were incubated for 1 h at 37 °C with JW8/5/13 cell supernatant, washed two times with PBS and incubated for 1 h at 37 °C with NIP-BSA (100 ng ml⁻¹) in pure M199/Ham’s F12 medium. The cells were then prepared for immunofluorescence staining with FITC-labeled anti-IgE. (B) Heat-aggregated IgG (30 µg ml⁻¹) was internalized for 15 min into THP-1 cells grown with 200 nM PMA for 2 d or P-STS cells grown for 3 d with 1 µg ml⁻¹ LPS, cells were fixed and prepared for immunofluorescence staining with FITC-labeled anti-IgG. (C) Immunofluorescence staining of Raji cells or Il-4 treated P-STS cells (both not permeabilized) with anti-CD23 (Alexa 488-labeled secondary antibody). (D) Expression of genes encoding IgE receptors assessed by real-time PCR analysis. Real-time PCR analysis was performed using the ΔΔCt method for relative quantification. Expression mRNA levels of the target gene were normalized to the average of the HKGs and calculated relative to the corresponding mRNA levels of Raji cells (equated to 1.0). Shown are genes encoding IgE receptors such as FCER1A (encoding the high-affinity receptor for IgE, alpha subunit) and FCER2 (also known as CD23, encoding the low-affinity receptor for IgE); additionally, CD19 is shown as the B-cell identity marker. Results are shown as mean values in one experiment ± SD and are representative of two independent experiments. *Note that FCER1A in Raji cells was classified as a low expressing gene in our analysis (30 < Ct < 35).