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To study the expression of src-related protooncogenes during the development of myeloid cells and the regulation of these genes by the colony-stimulating factors that control myelopoiesis, normal monocytic cells at distinct stages of differentiation were derived from murine bone marrow with the monocytic lineage colony-stimulating factor CSF-1. Protooncogene expression was also examined in uncultured human myeloid leukemia cells. While c-src transcripts were detected in myeloid leukemia cells representative of all stages of differentiation, the highly related gene c-fgr was expressed at high levels only at later developmental stages, both in normal cells committed to the monocytic lineage and in leukemic cells with a differentiated myelomonocytic phenotype. When bone marrow-derived monocytic cells were synchronized and stimulated to proliferate with CSF-1, c-fgr transcripts (but not transcripts from the highly related genes c-src or c-yes) were induced 8 hr after the addition of CSF-1 and decreased to low levels by 20 hr as the monocytic cells entered S phase. The selective induction of c-fgr mRNA by CSF-1 suggests that this tyrosine kinase may have a unique function in normal monocytic cells, distinct from other src-related tyrosine kinases.

Four‐color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE‐TR and PE‐CY5 (or PerCP). For two‐laser excitation APC is generally used in place of PE‐TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate. Cytometry (Comm. Clin. Cytometry) 38:161–175, 1999.  © 1999 Wiley‐Liss, Inc.

This unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two‐, three‐, and four‐color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis. Keywords: flow cytometry; immunophenotyping; indirect staining; direct staining; multicolor staining; immunophenotypic analysis.