differentiate in particular numbers. 



One of the ways in which cell differentiation 

 may be studied in these two slime molds is by 

 immunological methods; and in this seminar 

 today I want to talk about the use of fluorescent 

 antibody in studying differentiation. This par- 

 ticular method was first employed by Takeuchi 

 (1) in studies on Dictyostelium. This seminar is 

 based upon a study which has recently been 

 accepted for publication (2). 



The first step in doing an immunological 

 study involves the production of antisera (Table 

 I). Antibody was produced to three species or 

 strains of slime molds: D. discoideum, D, mu- 

 coroides (strain TYP) and a mutant of D. muco- 

 roides isolated and reported by Filosa (3), 

 These antisera were made to vegetative amoe- 

 bae, migrating pseudoplasmodia and mature 

 sorocarps, in each instance; that is, all three 

 stages were used in producing the antisera of 

 any one species. Now, the antiserum was con- 

 jugated with fluorescein iso-thiocyanate by more 

 or less conventional means, the salient points 

 of which involved the precipitation of gamma 

 globulin by cold methanol, weighing of a small 

 sample of the globulin solution with a micro- 

 balance in order to determine the total amount 



of globulins in the sample, and mixing the 

 globulins with 0.0188 mg of fluorescein per mg 

 of globulin (an amount we found to be optimum). 

 Following conjugation at 5^C for 15 to 18 hours, 

 the samples were centrifuged and then run 

 through a Sephadex column to remove nonin- 

 corporated fluorescein from the labeled globulin. 

 Such serum was used in staining various stages 

 of the slime molds. Unless otherwise indicated 

 homologous antiserum was used in the staining 

 procedure. 



Figure 2 shows D. mucoroides amoebae 

 removed from an aggregating stream. We find 

 that such cells, or such groups of cells, re- 

 moved from the stream will stain with various 

 intensities. Note the two extremes here: very 

 dark cells which stained with little intensity and 

 other cells which stained with a considerable 

 intensity. I believe these correspond to the so- 

 called 'Tjright" and "dark" cells which Takeuchi 

 (1) reported. I'll discuss the possible signifi- 

 cance of these cells later on. 



The early aggregates were sectioned at 

 about 5 microns. Although bright and dark cells 

 appear in the aggregating stream, once the cells 

 aggregate to form a cell mass in the early 

 aggregate the stain is more or less homogeneous 



TABLE I 

 Preparation of Conjugated Antisera ' 



1. Ganuna globulins precipitated from 1.0 volume serum by cold methanol. 

 Reagents and fractionation procedure described by Dubert _et ^. (8) . 



2. Globulins redissolved in 0.85 volumes of 1.0% NaCl . 



3. 100 ;ul aliquots of globulin solution dried and weighed, on Cahn ultra- 

 micro balance. Correction calculated for weight of NaCl in aliquot. 



4. Globulin solution dilutea with 0.15 volumes of 1.0 M carbonate- 

 bicarbonate buffer at pH 9.0. 



5. Globulin solution placed in 250 ml Erlenmeyer flask. Ice crystals 

 produced in globulin solution by immersing flask in dry ice-methyl 

 cellosolve bath (9) . 



6. In presence of ice crystals 0.0188 mg fluorescein iso-thiocyanate 

 added per mg globulin and mixed with magnetic stirrer at 5°C for 

 15-18 hours (10) . 



7. Centrifuged 20 minutes at 3000 X G in refrigerated centrifuge to 

 remove particulate niatter resulting from conjugation. 



8. Purification of f luorescein-conjugated globulins utilizing a G-25 

 fine Sephadex column (Pharmacia Fine Chemicals, Inc.) (11). 



' From Gregg, 1965 (2), reproduced with permission of Developmental Biology, published 

 by Academic Press. 



94 



