Fig. 3.

Microscopic and luminescence-based determination of detached cell and merozoite number, dependent on the parasite age in infected mosquitoes. a The relation of the parasite age in infected mosquitoes to the number of detached cells obtained and the relative merozoite number was investigated from day 16 to day 30. A critical cut off (red line), in detached cell numbers obtained is at 500 detached cells (4 × 10⁶ merozoites), as the success rate of detached cell transfections using fewer than 500 detached cells decreases. Seeding and infections were performed according to the present protocol, collected detached cells were counted using a fluorescent microscope at 65 hpi and merozoite number was determined using a luminescence based assay, n = 3. Error bars denote SD. b Determination of luminescence of detached cells obtained over time, using sporozoites from day 16 to day 30 post-blood feed. Detached cells of PbmCherry_Hsp70-NLuc parasites were obtained and collected according to the present protocol. Aside from quantification of detached cell numbers by fluorescence, luminescence was used to estimate the number of detached cells used for transfection from day 16 onwards, following mosquito feeds. Luminescence correlates with number of detached cells contained, and suggests that at days 16–23, the number of viable merozoites remains relatively constant, while a significant decrease in luminescence is identified from day 24 onwards (denoting less detached cells and less merozoites). Values shown are the result of three independent experiments repeated in triplicate. Error bars denote SD. c PbmCherry_Hsp70-NLuc detached cells were collected and disrupted to release merozoites. The total number of merozoites of a single detached cell was counted, and a limiting dilution was performed to achieve one merozoite, which was lysed and its luminescence measured. The radiance calculated was 9.9 × 10⁴ p s⁻¹ cm⁻². Performing a limiting dilution from 1 to 10⁶ merozoites we defined that a positive, significant (R² = 0.96) correlation exists between merozoite number and the log₁₀ of the relative light units of luminescence. This in turn allowed us to estimate the total number of merozoites in single individual detached cells and of the complete population of detached cells used for transfection. Values shown are the result of ten separate limiting dilutions from three independent experiments