Expression of FL in PBMC or BMMC. (A) Immunoblot analysis of FL. Recombinant human CHO-derived FL (rhFL; 5 ng; lanes 1 and 7) or cell lysates from 1 × 106 PBMC (lanes 2 through 4) or BMMC (lanes 8 through 10) from 3 healthy donors were analyzed by Western blotting with anti-FL MoAb M5. Controls: lanes 5 and 6, PBMC and rhFL, respectively, analyzed with rat IgG2a (control Ab); lanes 11 and 12, BMMC and rhFL, respectively, analyzed with M5 preincubated with 50-molar excess of rhFL before probing (M5 Ab-blocked). Migration of protein size markers is indicated; arrowheads point to 30- and 36-kD proteins. The amount of FL can be estimated as approximately 1 to 5 ng/106 mononuclear cells, because signals are weaker than those given by 5 ng of rhFL (lanes 1 and 7) and no signal is given by 1 ng of loaded rhFL (not shown). (B) Flow cytometric analysis of cell surface FL. PBMC or BMMC were stained with FITC-conjugated MoAb M5 (shaded area) or with FITC-conjugated control rat IgG2a (broken line). (C) Flow cytometric analysis of intracellular FL. PBMC or BMMC were preincubated with MoAb M5 to block cell surface FL, permeabilized with 0.1% saponin, and stained with FITC-conjugated M5 (shaded area). Control staining was with FITC-conjugated M5 preincubated for 30 minutes with a 100-fold excess of rhFL (solid line) or BSA (dotted line).
