Splenic Granulopoiesis in Mice following Administrationof Cyclophosphamide1

C57BL x DBA/2 F5mice bred in our laboratory from stock purchased from The Jackson Laboratory, Ban Harbor, Maine, were used in all experiments. Animals were between 8 and 15 weeks of age. Cyclophosphamide (100 mg) with sodium chloride (45 mg) (Mead Johnson Laboratories, Evansville, Ind.) was dissolved in 5 ml preservative-free sterile water and admin istered (200 mg/kg) i.p. Control mice were given injections ofa 0.9% NaCI solution. Suspensions of munine spleen cells were prepared by gentle dispersion through stainless steel sieves into CMRL culture media. Marrow cell suspensions were obtained by flushing the isolated mouse humerus with CMAL. Peniph eral blood was obtained by orbital sinus puncture. Cell concentrations were measured with an electronic particle counter (Model ZBI Coulten counter; Coulten Electronics, Inc. , Hialeah, Fla.) and from 200 cell differential counts of Wright's-stained blood smears and 500 cell differential counts of cytocentnifuged spleen and marrow cell suspen sions. The morphological features of granulocytes and their precursors in mice have been described previously(7). Briefly,proliferative c llsincludedmyeloblasts,pnomyelo cytes, and myelocytes, in which the nucleus has a central opening the diameter of which was less than one-half the diameter of the doughnut nucleus. Granulocytes with a widen nuclear central opening on segmented nucleus were defined as nonpnolifenative cells. In vitro,growthof CFU-Cfrommousespleenandmarrow was measured by using the soft gel system as described by Pluznik and Sachs (21) and Bradley and Metcalf (4). Meth ylcellulose (Dow Conning Corp. , Midland, Mich.) was used in placeof agar. Cellswere suspendedin 1.8% methylcel lulose, containing culture medium CMRL, 15% horse Se rum, and 10% L-cell-conditioned medium. The combined mixture was then plated in triplicate in 1-mI volumes in culturedishes35 x 10 mm and incubatedat37°in7.5% CO2 for 7 days. Colonies containing more than 50 cells were counted with an inverted microscope.