Table 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6	Parasitemia is too low to generate the LP and HP cultures	Parasitemia of the starting culture was too low, parasites proliferated poorly or many cells were lost during synchronization	Allow parasites to complete another IDC before returning to this step
13	Not enough cells are obtained after synchronization to seed the required number of 96-well plates	Parasitemia of the starting culture was too low, parasites proliferated poorly or many cells were lost during synchronization	Allow parasites to complete another IDC before returning to this step. Use fresh RBCs. Meanwhile, the CM can be stored at 4 °C for at least 90 d
23	Low parasite multiplication rate under noninduced conditions	Poor asexual proliferation. The RBCs may be too old or, for other reasons, do not support optimal parasite growth	Repeat the assay with a fresh batch of RBCs. It may be beneficial to combine RBCs from multiple donors for culturing
	Low parasite multiplication rate under CM-induced conditions	Note that parasite multiplication rate of CM-treated cultures typically drops by two- to threefold compared with parasites grown under noninducing conditions. This is indicative of a good induction of gametocyte production, rather than indicating a problem	If the parasite multiplication rate drops below 2, it may be necessary to further optimize the assay by using different CM conditions (e.g., lower the concentration of the CM working solution from 90% to 87.5%)
	Drug treatment induces a premature induction of the tdTom reporter at 28 ± 4 h.p.i.	Parasites show a ‘faux’ induction response to drug treatment (see ANTICIPATED RESULTS)	Perform gametocyte quantification at 90 ± 4 h.p.i. The transient signal from ‘faux’-induced parasites is not detectable anymore at this time point (see ANTICIPATED RESULTS)
25	CM treatment fails to induce gametocytogenesis	Small changes in assay conditions may drastically alter the rate at which gametocytes are produced. Whereas CM collected from cells at a parasitemia below 5.5% may lead to poor inductions, the application of other CM conditions may result in the complete killing of the experimental parasite population. Note that different stains may respond differently to CM treatment	We recommend performing an initial testing of the assay using CM working solutions at 90, 92.5 and 95% (vol/vol). This should allow achieving similar results to those shown in Figure 2. In the case of low gametocyte production, increase the concentra- tion of the CM working solution (e.g., from 90% to 95%). Alternatively, increase the parasitemia of the culture used to produce the CM (see Step 10). If CM treatment causes complete killing of the experi- mental cell population, lower the concentration of the CM working solution. Alternatively, lower the parasitemia of the culture used to produce the CM
	Many gametocytes are produced under noninducing conditions	Parasite culture conditions may have caused a self-induction of gametocyte production. Cells of the parasite strain used may convert to the sexual pathway at a high intrinsic rate (high baseline conversion)	Repeat the assay by strictly maintaining parasitemia of the experimental culture below 1% before seeding. Exchange the medium of this LP culture daily. Perform the assay with a cell line showing lower baseline sexual conversion
26	The tdTom-positive gametocyte population cannot be clearly distinguished from asexual (tdTom negative) parasites	Some gametocytes are too young and have not yet acquired enough tdTom reporter protein for detection	Perform flow cytometry to determine gametocytemia (Steps 24 and 25) at a later time point (e.g., 90 ± 4 h.p.i.)