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The 2 fold concentrated sera was obtained in an evaporator centrifuge (CentriVap concentrator-Labconco) at room temperature.
The fluorescence was compared to insects that ingested inactivated 2 fold concentrated human sera.
The fluorescent dye propidium iodide was used to access the death of epithelial cells exposed to the complement from the 2 fold concentrated human sera.
The fluorescent dye propidium iodide was used to access for the death of epithelial cells when exposed to the complement present in the 2 fold concentrated human sera.
Aliquots of crude cell extracts were diluted in 2 fold concentrated electrophoresis sample buffer containing SDS, heated and analyzed by SDS-polyacrylamide gelectrophoresissis (PAGE) [9], [10].
Starving fourth instar nymphs were forced to ingest 50 µL of 2 fold concentrated human sera containing 2.5 µg/ml of propidium iodide.
Figure 1-G shows groups of dead cells marked in red by the propidium iodide dye and, Figure 1-H shows the absence of marked areas in insects that ingested inactivated 2 fold concentrated human sera.
No marks were observed when insects were forced to ingest 50 µL of inactivated two fold concentrated human sera (Figure 1-F) except for the natural fluorescence similar to that in Figure 1-B.
With this apparatus, 50 µL of normal sera or 2 fold concentrated sera (active or inactivated by 30 min at 56°C) were injected into the digestive tract of the bug.
When the insects were forced to ingest two fold concentrated human sera, the inhibitors inside the midgut were not sufficient to prevent the intestinal epithelium from the complement attack.
Gelatin was added from a 10 fold concentrated stock containing pre-boiled gelatin type A (20 mg/ml, Sigma, Cat. # G 8150) and 1% SDS.
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