Main sol. 1514
Internal Solution-ID: S3133
1 Medium for maintenance of SF-9 cells (ACC 125) for cultivation of Rhabdochlamydiae. | |||||
Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
---|---|---|---|---|---|
Grace's Insect Medium
(Sigma Aldrich, G8142) |
900 | ml | - | - | |
Fetal bovine serum
(heat-inactivated, 56°C, 30 min) |
100 | ml | - | - | |
2 Filter-sterilize | |||||
3 Maintenance conditions of SF-9 cells (see also DSMZ catalogue for ACC 125): | |||||
4 SF-9 cells are cultivated at 25 - 29°C without the addition of CO2 in flasks (25 cm2) with tightly closed lids. When a confluent layer has formed, infection can be carried out. | |||||
5 Infection with Rhabdochlamydiae: | |||||
6 Exchange medium and add 500 – 1000 µl of EB stock solution (thawed quickly to 37°C). Centrifuge for 1 h onto the cell layer at 1600 rpm at 20°C. | |||||
7 Maintenance/Passage of infected insect cell cultures: | |||||
8 Infected cultures can be maintained e.g. in 25 cm2 flasks (e.g. Nunc flasks, VWR, 734- 2081) at 27°C in an incubator (no special requirements concerning CO2 or light). If the percentage of infected cells is still low (which might occur initially after inoculation of cultures with bacteria from a frozen stock) infected cultures may be maintained by weekly passage (1:5). Infection of yet uninfected cells by added bacteria or bacteria released from neighboring infected cells is facilitated by centrifugation (130 x g, 15 min) of the culture flask. In well-infected cultures host cell lysis will be visible (the bacteria cause host cell lysis within approximately 5-7 days after invasion of host cells). To maintain such cultures, bacteria have to be transferred by adding culture supernatant (approximately 0.5-1 ml) to fresh culture flasks containing uninfected insect cells (approximately semi-confluent culture) each 7-10 days. |
1514 | Grace's Insect Medium |