All C. parvum growth assays (aside from the initial metabolite screen) were performed as detailed here, with any modifications stated in the specific experimental sections. HCT-8 cells were plated at 1.5 × 105 cells per well in black-sided, optically clear-bottomed 96-well plates (Greiner BioOne) and grown for ~24h until confluent. Cells were infected with 1 × 105 bleached oocysts per well. After ~24h of infection/treatment, cells were fixed in 4% formaldehyde for 10 min, washed twice in DPBS, and then permeabilized and blocked in PBS containing 0.1% Triton X-100 and 1% BSA for 20 min before antibody staining. C. parvum parasites were labeled with Pan-Cp (rabbit polyclonal antibody raised against C. parvum that recognizes all stages of the parasite34 (link)) diluted 1:2,000 in PBS containing 0.1% Triton X-100 and 1% BSA, followed by Alexa Fluor goat anti-rabbit 488 secondary antibody (Thermo Fisher Scientific, diluted same as primary antibody). Host cell nuclei were stained with Hoechst 33342 (5 μg/mL, Thermo Fisher Scientific) for 20 min. Plates were imaged with a 10× objective on a BioTek Cytation 3 cell imager (9 images per well in a 3 × 3 grid). BioTek Gen5 software version 3.08 (Agilent) was used to quantify the total number of parasites (puncta in the GFP channel) and host cells (nuclei in the DAPI channel) per well.
Biotek gen5 software version 3
Manufactured by Agilent Technologies
BioTek Gen5 software version 3.08 is a data analysis and instrument control software. It provides functionalities for managing experimental data and controlling compatible laboratory equipment.
Automatically generated - may contain errors
2 protocols using biotek gen5 software version 3
1
Quantitative Cryptosporidium parvum Growth Assay
2
Quantifying Toxoplasma Infection in Host Cells
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HCT-8 cells were plated at 1 × 105 cells per well in black-sided, optically clear-bottomed 96-well plates (Greiner Bio-One) and grown until confluent. Cells were infected with 1 × 105 bleached wild type oocysts per well, and treated with 500 μM IAA or vehicle control for 48 h. After 48 hpi, monolayers were fixed in 4% formaldehyde for 10 min, washed twice in DPBS, and then permeabilized and blocked with TSS buffer for 20 mins. Parasites were labeled with rabbit PanCp diluted 1:2,000 followed by anti-rabbit Alexa Fluor 488. Host cell nuclei were stained with Hoechst for 20 min. Plates were imaged with a 10× objective on a BioTek Cytation 3 cell imager (9 images per well in a 3 × 3 grid). For measurements of CpGP-mAID-TIR1 parasite growth with or without IAA treatment, HCT-8 cells were infected with 5 × 104 bleached CpGP-mAID-TIR1 oocysts per well. Monolayers were treated with 500 μM IAA or vehicle control for 24 h or 48 h. After 24 hpi or 48 hpi, monolayers were fixed and stained as previously described. Plates were imaged with a 20× objective on a BioTek Cytation 3 cell imager (36 images per well in a 6 × 6 grid). BioTek Gen5 software version 3.08 (Agilent) was used to quantify the total number of parasites (puncta in the GFP channel) and host cells (nuclei in the DAPI channel) per well.
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