Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes: Maintenance of response potential of precursors and dissociation between proliferation and effector function of activated cytotoxic lymphocytes

1988

Cytometry

Self Cite

Several types of studies on regulation of cell activation and cycling have become possible as a result of the development of methods for rapidly measuring cell cycling activity (2,3,6-9). In addition to allowing direct quantitation of the frequency of S-phase cells, the fluorescence assay for bromodeoxyuridine (BUDR) incorporation has provided a means of quantitating the rate of DNA synthesis per cell, thus allowing an assessment of inhibitors of DNA synthesis on both normal lymphoblasts and tumor cells (6,7). The ability to quantitate total DNA, RNA, and cellular protein on a per cell basis allows for an assessment of the Go to GI (or GIa to Glb transition in lymphocyte activation and thus a n assessment of activation in the absence of cell cycling (see, e.g., 17). MATERIALS AND METHODSCells were pulsed with BUDR (Sigma Chemical Co., St. Louis, MO) by adding 1/10 volume of lo-* M BUDR directly to the cultures. After 30-60 rnin at 37"C, the cells were harvested, diluted five to tenfold in cold 0.15 M phosphate buffered saline (PBS; pH 7.2) and centrifuged through a cushion of calf serum. After two more washes in PBS (no serum), the cells were fixed at lo6 celldm1 in 70% ethanol (6) and held on ice for 30 min to 2 h. The cells were then centrifuged, resuspended in 0.1 ml of 0.85% NaCl and brought to 5 x lo5 cells/ml in 2 N HC1. Methods for measuring total DNA content by mithromycin stains and for measuring total protein by FITC stains have been described previously (2,6,7). A slightly modified version of the technique described by Traganos et al. (9) was used to quantitate total RNA content of cells. The ethanol-fixed cells were pelleted and resuspended to lo6 celldm1 in 0.15 M PBS, pH 6.0, containing 7 mg/ml sodium citrate and 5 x lOP3M EDTA. The cells were held at 37°C for 5 min, centrifuged, and resuspended to 4 x lo6 celldm1 in 0.1 N HC1. After 2-3 min at room temperature, a fourfold volume of PBScitrate buffer containing 5 ug/ml acridine orange was added. The cells were held for 30 min on ice before analysis. The fluorescence was excited by laser light at 488 nm. The fluoresced light was passed through (green) or reflected 90" by (red) a 560-nm dichroic mirror, The passed light was further filtered through a 530/30 bandpass filter and the reflected light was passed through a 620-nm longpass filter. RESULTS AND DISCUSSIONOur studies on the regulation of cell proliferation in vitro resulted in the observation that macrophages generated in vitro (MO-c) could, if properly activated, inhibit the proliferation (3H-thymidine incorporation) of tumor cells via a nontoxic and completely reversible cellcontact-dependent mechanism (4,5,10). The question that needed to be answered was whether the inhibition was due to failure of a major fraction of the cells to incorporate thymidine or was due to a reduced incorporation rate by all of the cells. Analysis of total DNA content was not satisfying (Fig. 1). Although there appeared to be a n increased frequency of cells in GI, the G1 peak was broader than control, and the pres...

show abstract

mentioning

confidence: 99%

Problems and applications of cell cycle analysis: Distinguishing G0 From G1 and G1 From S Phase

1988

Cytometry

Self Cite

show abstract

Functional characteristics of in vitro generated macrophages: A transient refractory state precedes reinducibility of a spatially restricted, possibly contact-dependent, cytostatic mechanism

1987

Cellular Immunology

Evidence for involvement of TNF-α in the induction phase and IFN-β in the effector phase of antiproliferative activity of splenic macrophages

1992

Cellular Immunology