Automated Synchronization of Plasmodium falciparum Parasites by Culture in a Temperature-Cycling Incubator
互联网
899
Continuously automated synchronization of Plasmodium falciparum malaria parasites can be achieved by culturing in a custom temperature-cycling incubator (TCI) whose process controller is programmed to periodically change between three different temperatures:
| 1. |
Shortly after invasion of merozoites into erythrocytes at about 37�C, the culture temperature is lowered to about 17�C, and the maturation of ring forms (Rgearly ?Rglate ) is suspended (and perhaps some remaining schizonts are killed). The time ramping down to and holding at 17�C, generally between 2.5 and 7.25 h, is determined by adding times for the next three steps below and subtracting from 48 hr. Thus, the total cycle length for any given parasite isolate is adjusted to 48 h, even though various free-cycle times at 37�C for different isolates range from about 38 to 45 h.
|
| 2. |
When the culture temperature is again raised to about 37�C, ring stages mature normally to trophozoites with pigment. Depending on the characteristics of the parasite isolate and its adaptation to culture, we allow between 12.75 and 18.5 h for this (including time for ramping to temperature).
|
| 3. |
When the culture temperature is raised to about 40�C, trophozoites (Tr) continue to mature up to a point (Trearly ?Trlate ), but then further maturation to schizonts (Trlate ?Sz) is blocked, so that parasites at earlier stages catch up to those at later stages. We allow between 9.5 and 10.5 h for this.
|
| 4. |
When the culture temperature is lowered again to about 37�C, maturation to schizonts (Trlate ?Sz) proceeds, and merozoites are released and invade erythrocytes. We allow between 18.5 and 22.5 h for this.
|
