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MB Sample ID: SA193568

Local Sample ID:	0424_PP_Cysten_T1_2
Subject ID:	SU002133
Subject Type:	Other organism
Subject Species:	Toxoplasma gondii
Taxonomy ID:	5811

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Sample Preparation:

Sampleprep ID:	SP002139
Sampleprep Summary:	Uninfected and infected myotubes were prepared. In both cases, two T150 dishes were pooled into one sample. For bradyzoite samples, myotubes were infected with 3.2106 Pru-tdTomato tachyzoites corresponding to a MOI of 0.3 and cyst formation was induced for indicated time. On the day of harvest, infected samples and uninfected host cell controls were placed on ice, medium was removed and monolayers were washed three times with ice-cold PBS. Cells were then harvested by scraping into 10 ml ice-cold 0.05 % BSA in PBS per T150 dish. Cysts were released from the monolayer via forcing through a 23G needle (Sterican®) 25 times with a syringe and collected via centrifugation (1,200 x g, 10 min, 0 °C). The supernatant was removed, the pellet was resuspended carefully in ice-cold 2 % BSA in PBS containing 200 µl DBA-coupled beads (preparation described below) and samples were incubated for 1 h at 4 °C with gentle shaking. Subsequently, the samples were placed in a magnetic stand on ice, washed five times with 0.1 % BSA in PBS to remove cell debris, followed by two washing steps with PBS to remove residual BSA. Cysts and beads were then collected via centrifugation (1,200 x g, 10 min, 0 °C), shock frozen in liquid nitrogen and stored at -80 °C until extraction. Tachyzoite samples were generated in T150 dishes by infecting myotubes and HFF cells with 3.2107 tachyzoites corresponding to an MOI of 3 for 48 h. Medium was replaced by ice-cold PBS and monolayers were scraped and passaged through a 27G needle. Tachyzoites were filter-purified through a 3 µm filter (Whatman) and PBS-washed by centrifugation (1,200 x g, 10 min, 0 °C) three times. All samples were extracted simultaneously in 80 % acetonitrile for LC/MS analysis as described below. Bead-only controls were processed equally. Bead-supplemented tachyzoite controls were processed equally to cyst samples, replacing washing steps via magnetic stand by centrifugation (1,200 x g, 10 min, 0 °C). Preparation of beads Coupling of DynabeadsTM MyONETM Streptavdin T1 (Thermo Fisher Scientific) to DBA was done as described in the manufacturer's protocol. Briefly, 200 µl beads/sample were resuspended in 1 ml PBS by vortexing, washed three times with PBS in a magnetic stand and resuspended in 1 ml PBS containing 50 µg DBA / sample. The tube containing the DBA-magnetic bead mixture was incubated on a rotary mixer for 45 min at RT. Uncoupled DBA was removed by washing the coated beads three times with PBS. After washing, the DBA-coated beads were resuspended in 2 ml PBS containing 2 % BSA.

Sampleprep ID:

SP002139

Sampleprep Summary:

Uninfected and infected myotubes were prepared. In both cases, two T150 dishes were pooled into one sample. For bradyzoite samples, myotubes were infected with 3.2*106 Pru-tdTomato tachyzoites corresponding to a MOI of 0.3 and cyst formation was induced for indicated time. On the day of harvest, infected samples and uninfected host cell controls were placed on ice, medium was removed and monolayers were washed three times with ice-cold PBS. Cells were then harvested by scraping into 10 ml ice-cold 0.05 % BSA in PBS per T150 dish. Cysts were released from the monolayer via forcing through a 23G needle (Sterican®) 25 times with a syringe and collected via centrifugation (1,200 x g, 10 min, 0 °C). The supernatant was removed, the pellet was resuspended carefully in ice-cold 2 % BSA in PBS containing 200 µl DBA-coupled beads (preparation described below) and samples were incubated for 1 h at 4 °C with gentle shaking. Subsequently, the samples were placed in a magnetic stand on ice, washed five times with 0.1 % BSA in PBS to remove cell debris, followed by two washing steps with PBS to remove residual BSA. Cysts and beads were then collected via centrifugation (1,200 x g, 10 min, 0 °C), shock frozen in liquid nitrogen and stored at -80 °C until extraction. Tachyzoite samples were generated in T150 dishes by infecting myotubes and HFF cells with 3.2*107 tachyzoites corresponding to an MOI of 3 for 48 h. Medium was replaced by ice-cold PBS and monolayers were scraped and passaged through a 27G needle. Tachyzoites were filter-purified through a 3 µm filter (Whatman) and PBS-washed by centrifugation (1,200 x g, 10 min, 0 °C) three times. All samples were extracted simultaneously in 80 % acetonitrile for LC/MS analysis as described below. Bead-only controls were processed equally. Bead-supplemented tachyzoite controls were processed equally to cyst samples, replacing washing steps via magnetic stand by centrifugation (1,200 x g, 10 min, 0 °C). Preparation of beads Coupling of DynabeadsTM MyONETM Streptavdin T1 (Thermo Fisher Scientific) to DBA was done as described in the manufacturer's protocol. Briefly, 200 µl beads/sample were resuspended in 1 ml PBS by vortexing, washed three times with PBS in a magnetic stand and resuspended in 1 ml PBS containing 50 µg DBA / sample. The tube containing the DBA-magnetic bead mixture was incubated on a rotary mixer for 45 min at RT. Uncoupled DBA was removed by washing the coated beads three times with PBS. After washing, the DBA-coated beads were resuspended in 2 ml PBS containing 2 % BSA.