A Micromethod for Lymphoblastic Transformation of Mouse Lymphocytes from Peripheral Blood

Abstract

Although several micromethods for culturing human peripheral lymphocytes exist (1–3), attempts to develop similar procedures for small laboratory animals have not been equally successful (4–6). First, mouse lymphocytes appear less responsive to PHA stimulation than human lymphocytes under their respective culture conditions (7, 8). Second, because cell suspensions from whole organs have generally been used from mice, direct comparisons to human peripheral lymphocytes are difficult; also, the mice had to be killed, thus eliminating them from further use or breeding experiments. Third, the culture systems for mouse lymphocytes employed varying amounts of heterologous serum (5, 6) or lacked appropriate standardization with respect to optimal culturing conditions and culture time. The lack of a fast and reproducible micromethod for lympho-blastogenic transformation in mice is regrettable because it precludes studies on the genetic control of transformation and initiation of cell proliferation without sacrificing the animals.

We now describe a reliable method for culturing mouse peripheral lymphocytes that (a) requires only 50 μl of blood, thus allowing the preparation of several cultures from individual mice without interference with their metabolism or reproduction, (b) avoids the need for tedious cell separations, thus retaining erythrocytes in culture which may be beneficial for successful blastogenic transformation (9), and (c) excludes the use of heterologous sera. The method should be useful in investigations of the function of peripheral lymphocytes and allow for study of their genetic control including linkage studies for the genes governing their response to PHA or other antigens.

Materials and Methods. The following strains of mice were used, AKR/J, C57BL/10ScSn, C57BL/10J, and C57L/J; unless stated otherwise, they were 10 ± 2-wk-old females.

Phytohemagglutinin-M (PHA-M) was obtained from Difco Laboratories, and culture media consisting of Trowell's T-8 and Ham's F10, TC 199, McCoy's 5a modified, Way-mouth's MD705/1, and RPMI 1603, 1629, 1630, 1634, and 1640 (10, 11), were purchased from Grand Island Biological Laboratory (Gibco).