Preparation of Antisera Specific for Guinea Pig B Cells

Abstract

Heterologous antisera against guinea pig B lymphocytes have been prepared in rabbits. Sera, obtained 1 week after a third injection of cells from a guinea pig leukemic B cell line (L₂C), were absorbed with guinea pig and sheep erythrocytes and guinea pig thymocytes. In the presence of complement, selected antisera killed both L₂C cells (85%) and B cells from Hartley outbred guinea pigs (85%). Normal rabbit serum kills guinea pig thymocytes and this ability was enhanced in the sera obtained from rabbits immunized with L₂C cells. After three absorptions with thymocytes the antisera retained their cytotoxicity for B cells but did not kill guinea pig thymocytes or peripheral T lymphocytes. The antigenic determinants on the L₂C cells that simulate anti-B cell activity are unknown but surface membrane immunoglobulins were excluded. The development of methodology for obtaining a potent and specific anti-guinea pig B cell serum should significantly improve the usefulness of guinea pigs as animals for in vitro studies.

Distribution of a Guinea Pig Lymphocyte Cell Surface Antigen of Defined Function, as Detected by the Mouse Monoclonal Antibody MSgp 1

“…(4) The la-bearing guinea pig B cell leukaemia EN-L2C, which bears cross-reacting antigens with peripheral guinea pig B cells [6], was used to screen for both la and B cell activity. The line was maintained by serial transplantation in strain 2 guinea pigs and re covered from the blood employing a gradient consisting of 3.15% Ficoll and 3.12% Hypaque (Pharmacia) in a I : I ratio.…”

E-Rosetting of Guinea Pig Lymphocytes with Lyophilized Rabbit Red Cells after Treatment with Papain and Glutaraldehyde

“…They were fed on GC 3 (Clea Japan Inc., Tokyo) until use a week later. Cell suspensions were made of their thymus, spleen and lymph nodes, as described previously [22,23). For a peripheral lymphocyte preparation blood was drawn into a heparinized syringe by cardiac puncture of the animals under anesthesia, diluted 3 times in Dulbecco's phosphatebuffered saline containing Mg2+ and Ca2+ (PBS) and then subjected to centrifugation on Ficoll-Hypaque of specific gravity 1.090.…”

“…Leukemic cells were harvested and separated on Ficoll-Hypaque from periph eral blood of moribund animals, as described previously [23].…”

“…A lymphoid population enriched for B-cells was prepared from cervical lymph nodes of Hartley strains, as described before [23].…”

“…As previously described [23] E-rosetting was performed in small test tubes by reacting 0.1 ml of a cell suspension containing 4x10'' viable lymphoid cells with 0.2 ml of a 0.8% suspension of rabbit red blood cells, held at 37°C for 15 min and centrifuged at 500 rpm (RavoI = 11.5) for 5 min at 4°C. Then the reaction mixtures were al lowed to remain pelleted in ice for a further 60 min until they were examined for the number of E-rosettes after addition of acridine orange and ethidium bromide under the epi-fluorescent micro scope equipped with a phase-contrast apparatus.…”