Expression of ALT proteins in L. mexicana cultured promastigotes. (A-D) Immunofluorescent detection of transgenic protein. Paraformaldehyde-fixed wild-type promastigotes were incubated with (A) anti-L. mexicana serum or (B) anti-ALT-1 antibody. (C) Cloned alt-1 transfected and (D) alt-2 transfected fixed parasites were incubated with an anti-ALT-1 antibody, which binds to both ALT-1 and ALT-2, and detected with an anti-mouse-FITC secondary antibody. (E-H) FACS analysis of transfected promastigotes of L. mexicana. Fixed promastigotes were stained, without permeabilization, with anti-ALT-1 antibody and analysed by FACS. (E) alt-1-transfected L. mexicana; (F) alt-2-transfected L. mexicana; (G) wild-type L. mexicana. Positive cells are those within the gate (M1) set above the threshold defined by reactivity of transfected parasites stained with normal mouse serum (H). (I, J) Multiplication of transfectants within macrophages. (I) In vitro-transformed wild-type amastigotes or (J) alt-1 transfected amastigotes were added to bone marrow-derived macrophages from CBA mice at a ratio of 10 parasites per macrophage. After 24 hours at 34°C the infected macrophages were observed by microscopy.