126.96.36.199 Infection of primary honey bee ventricular cells
- Isolate pupal gut cells from 10 days old pupae as described in detail in the BEEBOOK paper on cell culture techniques (Genersch et al., 2013).
- Transfer approximately 5E+7 Nosema spp. spores in AE-buffer (Qiagen) into chamber slides (2 well glass slide, VWR)
- Air dry for 3 hours at room temperature.
- Initialize spore-germination with 50 µl 0.1M Sucrose in PBS-buffer (BDH, Laboratory Supplies).
- Immediately resuspend 500 µl primary cell suspension in fresh L15 medium ( 1.49 % L-15, 0.4 % Glucose, 0.25 % Fructose, 0.33 % Prolin, 3% Sucrose, all w/v, pH 7.2; for a recipe see Table 2) with the germinating spores
- Incubate the spore-cell suspension at 33°C for 20 min.
- Add 1 ml of fresh and pre-warmed (37°C) BM3 medium (L 15 medium + 0.075 % Pipes, 3 % inactivated FCS, 1.2 % Yeastolate, 10 % antimycotic/antibiotic solution from Sigma-Aldrich, pH 6.7; for a recipe see Table 2).
- Long time incubation is performed in a cooling incubator (Thermo Fisher) at 33°C for 144 hours.
- Remove the supernatant by aspiration
- Fix cells with 500 µl of 4 % formalin solution (Roth) for 24 hours.
- Identify infected
cells by microscopic analysis (Figs 7A & B).
Although the infection of primary pupal cells can be achieved, this approach is time consuming, does not easily lead to reproducible results, and is accompanied by the problem of seasonal dependency because sufficient numbers of pupae are only available during the brood rearing period.
BM 3 medium pH 6.7
1000 ml L 15 medium + 0.75 g Pipes, 30 ml FCS (inactivated), 12 g Yeastolate
L 15 medium pH 7.2
14.9 g L-15 powder, 4.0 g
glucose, 2.5 g fructose,
Fig. 7 A-C. Infection assay with Nosema spp. and primary pupal gut cells: A. infected primary ventricular cell; B. dissolving primary cell releasing new spores; C. in situ-hybridization (ISH) of infected ventricular cells, infected cells are stained blue. Specific hybridization was performed with 16S rRNA (SSU) probes coupled with digoxigenin and subsequent colour reaction to detect hybridized probes. Bars (A, B) represent 10 μm and bar in C represents 50 μm.