animal heterokaryons

796

Cell Biology

DOUBLE

International

LABELLING

Reports,

OF PLANT/ANIMAL

Vol. 4, No. 8, August

1980

HETEROKARYONS

M.Ward', M.R.Davey', E.C.Cocking', R.H.Clothier$ M.Balls' and J.A.Lucy3. 'Department of Botany, and 'Department of Human Morphology, University of Nottingham NG7 2RD. 3Department of Biochemistry, Royal Free Hospital School of Medicine, 8 Hunter Street, London WClN 1BP. Twenty-four hours after subculture, Xenopus cells phagocytosed particles of carmine (0.02% w/v) added to the culture medium. After a further 24 hr the carmine was removed and fresh culture medium added. The Fenopus cells proliferated during the final 24 hr prior to harvesting for fusion (when over 70% clearly contained red granules). Xenopus cell fusion with carrot protoplasts was stimulated by protease pretreatment followed by incubation at high pH, high Ca ion concentration and raised temperature for 15 The interkingdom heteromin'. karyons were recognised by light microscopy as being similar in appearance to carrot protoplasts (which cannot phagocytose carmine particles) hut containing carmine particles introduced by the animal partner (Fig.la,c). Samples were incubated with rabbit antiserum directed against whole washed and then Xenopus cells, incubated with sheep anti-rabbit immunoglobulin antibodies conjug-

fluorescein isothiocyanate. cells were fluorescently labelled by this method, while there was no cross-reaction with unfused carrot protoplasts. Heterokaryons, similar in appearance to carrot protoplasts under bright field illumination, showed surface fluorescence under UV illumination. Immediately after fusion the fluorescence observed on heterokaryons was restricted to a limited area (Fig.lb), but after a few hours had spread to cover the whole heterokaryon (Fig. reflecting the movement of la), Xenopus specific components within the membrane. 68 of 111 heterokaryons identified by the innnunofluorescent method were also labelled with carmine, whereas 89 of 95 heterokaryons containing carmine also showed surface fluorescence carmine labelling promises to Thus, be a reliable and useful way of selecting viable Xenopuslcarrot heterokaryons. ' Ward et aZ. (1979) Somat. Cell 5, 529-536. Genet.

Xenopus/carrot Fig.1. karyons labelled with c=carmine particles. after fusion, bright illumination x 500.

(b) Same field of view as (a), UV illumination x 500. (c) 3f hrafter fusion, bright field illumination x 500. (d) Same field of view as (c), UV illumination x 500.

0309-1651/60/060796-01/$02.00/0

heterocarmine, (a) 4 hr field

ated

with

Xenopus

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