Tamoxifen was administered to P5 Tns3flox; Pdgfrα-CreERT; Rosa26stop-floxed-YFP and Tns3flox; Pdgfrα-WT; Rosa26stop-floxed-YFP littermates. Brains were dissected out at P7 in order to MACSort OPCs using an anti-PDGFRα antibody coupled to magnetic beads. OPCs were plated in poly-l -ornithine (Sigma, P4957)-coated µ-Slide 8 Well Glass Bottom slide (ibidi, 80827) at 40,000 cells/mm2 in OPC proliferative medium: DMEM/F12 (Life Technologies, 31331028), 5 mM HEPES buffer (Life Technologies, 15630056), 0.6% glucose (Sigma, G8769), 1× penicillin/streptomycin (Life Technologies, 15140122), N2 supplement (Life Technologies, 17502048), B27 supplement (Life Technologies, 17504044), 20 ng/µl EGF (PeproTech, AF-100-15), 10 ng/µl FGF-basic (PeproTech, 100-18B), 10 ng/µl PDGF-AA (PeproTech, 100-13A), and 20 μg/ml insulin (Sigma, I6634). After 3 days of proliferation, medium was replaced by growth factor-depleted medium. Cell differentiation was tracked for 3 days using time-lapse video recording. Cells were put in to a videomicroscope (Zeiss AxioObserver 7, provided by ICM-quant and CELIS facilities) with a humidified incubator at 37°C with a constant 5% CO2 supply. Images for both FITC and bright field were acquired every 10 min.
Slide 8 well glass bottom slide
Manufactured by Ibidi
Sourced in Germany, United States
The µ-Slide 8 Well Glass Bottom slide is a laboratory equipment product designed for cell-based assays. It features eight individual wells with a glass bottom. The slide provides a platform for culturing and observing cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Hepes buffer, by Thermo Fisher Scientific (2 mentions)
Basic fgf, by Thermo Fisher Scientific (2 mentions)
Pdgf aa, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Axio observer 7, by Zeiss (2 mentions)
Insulin, by Merck Group (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
P4957, by Merck Group (1 mentions)
Af 100 15, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Lab tek chambered coverglass systems, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
4 protocols using slide 8 well glass bottom slide
1
Tamoxifen-induced OPC differentiation
2
OPC Differentiation Tracking Protocol
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Tamoxifen was administered to P5 Tns3 flox ; Pdgfrα-CreER T ; Rosa26 stop-floxed-YFP and Tns3 flox ; Pdgfrα-WT; Rosa26 stop-floxed-YFP littermates. Brains were dissected out at P7 in order to MACSort OPCs using an anti-PDGFRα antibody coupled to magnetic beads. OPCs were plated in poly-L-ornithine (sigma, P4957) coated µ-Slide 8 Well Glass Bottom slide (Ibidi, 80827) at 40,000 cells/mm2 in OPC proliferative medium : DMEM/F12 (life technology, 31331028), 5mM HEPES buffer (life technology, 15630056), 0.6% glucose (Sigma, G8769), 1x penicillin/streptomycin (life technology, 15140122), N2 supplement (life technology, 17502048), B27 supplement (life technology, 17504044), 20ng/µl EGF (Peprotech, AF-100-15), 10ng/µl FGF-basic (Peprotech, 100-18B), 10ng/µl PDGF-AA (PeproTech, 100-13A) and 20μg/ml Insulin (Sigma, I6634). After 3 days of proliferation, medium was replaced by growth factor depleted medium. Cell differentiation was tracked for 3 days using time-lapse video recording.
Cells were put in to a videomicroscope (Zeiss AxioObserver 7, provided by ICM-quant and CELIS facilities) with a humidified incubator at 37°C with a constant 5% CO2 supply. Images for both FITC and bright field were acquired every 10 minutes.
Cells were put in to a videomicroscope (Zeiss AxioObserver 7, provided by ICM-quant and CELIS facilities) with a humidified incubator at 37°C with a constant 5% CO2 supply. Images for both FITC and bright field were acquired every 10 minutes.
3
Quantifying Parasite Protein Export Efficiency
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All fluorescence imaging was performed with a Zeiss AxioObserver Z1 epifluorescence microscope equipped with a Zeiss AxioCam MRm camera (Zeiss, Oberkochen, Germany). Tagging and knockout mutants were passed through the parasite life cycle and recorded at different time points. Asexual blood stages were imaged from peripheral blood or ex vivo blood cultures. Oocysts were recorded on days 10, and sporozoites on day 21 after the blood meal. Huh7 cells were seeded onto µ-slide 8 well glass bottom slides (Ibidi, Martinsried, Germany), infected with 10,000 sporozoites at subconfluence, and imaged 24, 48, and 72 hours after infection. Nuclei were visualized with Hoechst 33342 nuclear dye (1:1,000).
Export of EMAP1 was assessed by quantifying the raw integrated density (RID) of EMAP1-mCherry associated with the parasite and with the entire infected erythrocyte. Export efficiency was calculated as follows:
Export of EMAP1 was assessed by quantifying the raw integrated density (RID) of EMAP1-mCherry associated with the parasite and with the entire infected erythrocyte. Export efficiency was calculated as follows:
4
Culturing and Transfecting HeLa Cell Lines
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HeLa Kyoto (RRID:CVCL_1922) and HeLa rCDS cells (24) were cultured at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Biochrom), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell line identity of HeLa Kyoto cells has been authenticated using STR profiling (Promega PowerPlex 21 Kit; carried out by Eurofins Genomics, Ebersberg, Germany). Cell lines were grown from mycoplasma-free liquid nitrogen stocks. Passaged cells in culture in the lab are monitored regularly (every 4 months) for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Rockland, USA), and cell lines used here were contamination-free. For microscopy experiments, cells were seeded on slides within eight-well Lab-Tek chambered coverglass systems (Thermo Scientific, Waltham, USA) or Ibidi µ-Slide 8 Well Glass bottom slides (Ibidi, Planegg, Germany). At about 50% confluence, cells were transfected using Turbofect (Thermo Scientific, Waltham, USA) according to the manufacturer's instructions. 0.5 or 0.75 µg DNA was transfected per Labtek or Ibidi well, respectively. Tagged pCHIV derivatives were transfected in an equimolar ratio with their non-labeled counterpart. At 4 hpt, the transfection mixture was replaced by fresh medium.
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