Blast Transformation of Lymphocytes from Guinea Pigs, Rats, and Rabbits Induced by Mercuric Chloride in Vitro

The animals used were inbred Hartley strain guinea pigs (400-500 g), inbred Sprague strain rats (300400 g), and a purebred Dutch rabbit (1,300 g). Lymphocytes were obtained from the guinea pig and rat lymph nodes and the rabbit spleen. The animals were anesthetized with ethyl ether and exsanguinated via caridac puncture. The blood was defibrinated, and the serum was collected and filtered through a Swinnex filter with a mean pore diameter of 0.45 (Millipore Filter Corp., Bedford, Mass.). The lymph nodes or spleen were removed, trimmed of excess tissue, and placed in medium 199 (Microbiological Associates, Inc., Bethesda) containing 100 g of streptomycin and 100 units of penicillin per ml and 1% of a 200 mmole solution of L-glutamine. The lymphoid tissue was gently teased apart in this medium and filtered through a sterile column packed with a fine-mesh nylon. The resulting cell suspension was washed three times with this medium by centrifugation. The final cell button was suspended in the above-mentioned medium containing 20% filtered autologous serum to give a final cell density of 2.5-3.0 X 106 leukocytes per ml (>94% lymphocytes). The cell suspension was distributed in 2.5 ml volumes in 16X 125-mm capped plastic culture tubes (Falcon Plastics, Division of B-D Laboratories, Inc., Los Angeles). Several concentrations of HgCI 2 dissolved in a physiological saline solution were sterilized by filtration and added in 0.1-ml volumes to appropriate cultures. Triplicate or duplicate cultures were incubated at 37°C and harvested daily for a period of 6 days. Colcemid (0.25 g/ml) was added for the final 2 hr to induce metaphase mitotic arrest. Viability counts were made with the trypan blue dye-exclusion method (3). At harvest time, the cell suspensions were transferred to 5-ml conicalshaped centrifuge tubes, and the cells were deposited by centrifugation. The supernatant was discarded and the cell button was suspended in a drop of serum and smears were made. Lymphocyte transformation was assessed morphologically in May-Gr/inwaldGiemsa-stained smears by counting of lymphoblasts and mitotic figures in at least 2,000 mononuclear cells. Lymphoblasts were recognized by accepted criteria as large mononuclear cells exceeding 15 in diameter with deeply basophilic, often vacuolated, nongranular cytoplasm surrounding a large nucleus with homogeneously stained chromatin and often containing one or more nucleoli (4, 5). Transformation of the guinea pig lymph node cells was also measured by their uptake of tritiated thymidine (6). One pc of tritiated thymidine (specific activity of 6.7 c/mmole, New England Nuclear Corp., Boston) was added for the final 3 hr; at harvest, DNA synthesis was arrested by the addition of 0.05 ml of 1.20 M sodium azide, and the cultures were transferred to an ice bath (7). The cells were washed with 10 ml of cold physiological saline and deposited by centrifugation at 1,500 rpm for 8 min. The supernatant was discarded and 0.05 ml of 30% hydrogen peroxide was added for bleaching any red blood cells present. Decoloration occurred rapidly, and 30 sec later the cell suspension was washed three times in a cold physiological saline by centrifugation, and the cell button was dissolved in 1.0 ml of NCS solubilizer (Nuclear-Chicago Corporation, Des Plaines). 10 ml of a phosphor solution containing 4 g of PPO and 0.05 g of POPOP/liter' of toluene was added; the suspension was allowed to stand in the dark for 24 hr and its radioactivity then was measured in a Nuclear-Chicago series 720 liquid scintillation counter. The counts per minute were corrected for chemical and color quenching by the channels ratio