Surface expression of functional TfRs in erythroid and nonerythroid cell types.

Cells were immunolabeled with the chicken TfR-specific antibody JS-831 and a secondary FITC-conjugated antibody immediately after harvesting. The cells were subjected to flow cytometry (FACScan; Becton Dickinson) and results analyzed using the CellQuest software package. The histograms represent the frequency distribution of fluorescence intensity (FL-1 height) directly correlating with the TfR expression levels on the cell surface. As controls, cells were analyzed without staining, stained with an unspecific antibody (51/3) and FITC or solely with FITC. The background fluorescence was less than 6 units (not shown). The nonerythroid cell types CEF, LMH, and MC29-HD11 and the hematopoietic v-Ski–expressing progenitors were cultured under standard conditions (without additional iron supply; labeled −Tf). The erythroleukemic HD3E22 cells and the primary erythroid SCF progenitors were cultured either under physiologic iron supply (1 mg/mL Tf; for details see “Materials and methods”; labeled +Tf), or without additional iron source (−Tf) for at least 24 hours. CEF indicates chicken embryo fibroblasts; LMH, leghorn male hepatoma cells; MC29, MC29-HD11 macrophagelike cells; v-Ski, hematopoietic cells expressing the v-Ski proto-oncogene; SCF, SCF progenitors; HD3, HD3E22 erythroblasts.