Table 1.

Troubleshooting table

STEPS	PROBLEM	POSSIBLE CAUSE	SOLUTION
1–4	Low yield of parasites after Percoll purification	Parasitemia of culture is too low	Use culture with high parasitemias (normally 0.5–1% is enough)
		35% Percoll or culture suspension is added too fast, which strongly disturbs the 35/65 interface	Lean the pipette tip against the wall of tube and reach to the liquid surface and slowly add the 35% Percoll solution
6–9	Too much cell debris in the Percoll purification	Parasites culture suspension is added too fast, which strongly disturbs the 35/65 interface	Slowly add culture suspension to the 35% Percoll surface
10	The flow in the sorter is blocked	Cell aggregation occurs when cell concentration is too high	Lower cell density, complete separate cells by gently vortexing before sorting
11–13	Percentage of singly infected cells is low	Gating is not appropriate. You will get more doubly or triply infected RBCs or many uninfected cells and debris if the fractions chosen for sorting are higher or lower than the correct gate, respectively.	Pre-sorting is an option to seed cells on the glass slide, which are checked. Normally lower fractions are preferred though one might get more cell debris or uninfected RBCs
15–16	Single cell is not loaded into each well.	Plate is not placed in the right position	Make sure the plate is put on the receiving tray stand and reaches the end of frame
	RBCs undergo lysis during culture within two weeks	RBCs are not fresh when put into plate	Use fresh RBCs (not more than one week) Add new RBCs at 0.5% hematocrit to each well when changing medium on day 5 and 10