IMMUNOLOGICAL COMPETENCE OF SMALL LYMPHOCYTES 239 



low temperatures. From an initial 7 ml. of heparinized blood, 

 0'5-0'8 ml. of lymphocyte-erythrocyte suspension, containing 

 about I million lymphocytes/o-os ml., was obtained. The 

 preparation may be concentrated up to about 1-5 million 

 lymphocytes/ 0' 05 ml. by centrifugation and removal of plasma, 

 but preparations too heavy with cells were not well tolerated by 

 newborn mice following intracardiac injection. With certain 

 strains of mice, red cell contamination was greatly reduced by 

 sedimentation from the fdtered blood with polyvinylpyrrolidone 

 according to the method of Walford (i960). Alsever's solution 

 rather than heparin as anticoagulant gave better separation when 

 polyvinylpyrrolidone was used. Unfortunately, for unknown 

 reasons, several modifications of the latter procedure did not work 

 well with C57BL/6 blood. We were not able to obtain good yields 

 of viable mouse lymphocytes by various dextran-sedimentation 

 procedures (cf. Skoog and Beck, 1956; Walford, i960). However, 

 we have recently obtained pure and concentrated small lympho- 

 cyte preparations after sedimentation of red cells from filtered 

 blood by treatment with fibrinogen (Skoog and Beck, 1956). 



Lymphocyte counts of purified preparations were made in 

 Neubauer chambers in the usual manner. For differential counts, 

 Wright-stained smears were prepared and thoroughly examined 

 for contamination with leucocytes other than lymphocytes. 

 Erythrocyte contamination was not considered, since donor 

 erythrocytes were assumed to be immunologically null ui the 

 graft-versus-host assay. Cell viability was assessed by eosin dye 

 exclusion (Hanks and Wallace, 1958) employing i per cent 

 Eosin-Y. Final preparations were 96-100 per cent pure and 99-100 

 per cent viable with respect to small lymphocytes. After some 

 experience with the procedure, many preparations were obtained 

 that showed no leucocytes other than small lymphocytes among 

 hundreds of cells scored. The purity is directly, and the yield of 

 lymphocytes inversely, proportional to the amount of glass wool 

 employed in filtration. 



