Dictyostelium Growth and Fractionation

Cell culture was done following Brock et al. (Brock et al., 1999 (link)) in HL5 medium (Formedium Ltd, Norwich, England) using wild-type Ax2 cells, aprA⁻ strain DB60T3-8 (Brock and Gomer, 2005 (link)), and crlA⁻ strain JH557 (Raisley et al., 2004 (link)). Conditioned growth and starvation media (CM) were prepared and concentrated, and PBM buffer was made, following Brock et al. (Brock et al., 2002 (link)). Size fractionation was carried out as described in (Brock and Gomer, 2005 (link)). Western blots of fractions were stained with anti-AprA antibodies as described in (Brock and Gomer, 2005 (link)). To examine the size of the CfaD complex secreted by NC4 cells, 1×10⁶ NC4 cells were grown with live Klebsiella bacteria in PBM in a shaking suspension culture. As a control, bacteria were grown without Dictyostelium cells. After 36 hours, the Dictyostelium cell density was ~3×10⁷ cells/ml, and the supernatant was clarified and used for gel filtration. Photography of aggregates and fruiting bodies was performed as described in Brock et al. (Brock et al., 2002 (link)). Proliferation assays, calculation of doubling times, staining of nuclei, spore counts and spore viability assays were done as described in (Brock and Gomer, 2005 (link)).