For tissue-level analysis, one-fourth of a mouse brain was placed in 1 mL Trizol (Ambion), mechanically homogenized using 1 mm zirconia/silica beads (Biospec) for 30 seconds using a Mini-BeadBeater 16 (BioSpec). For gene expression analysis of magnetically-enriched cells, cells were homogenized in Trizol by pipetting. RNA was extracted from Trizol according to manufacturer’s instructions (Invitrogen). High Capacity Reverse Transcription Kit (Applied Biosystems) was used to generate cDNA. Quantitative PCR was performed using 2X Taq-based Master Mix (Bioline) and TaqMan gene expression assays (Applied Biosystems), or custom primers (Integrated DNA Technologies), run on a CFX384 Real-Time System thermocycler (Bio-Rad Laboratories). Murine Hprt and T. gondii Act1 were used for normalization for analyzing host and parasite gene expression, respectively, and relative expression is reported as 2(−ΔΔCT). The following Thermo Fisher mouse gene probes were used: Stat1 (Mm00439518_m1), Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Nos2 (Mm00440502_m1), Il6 (Mm00446190_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), Tnfa (Mm00443258_m1), Ccl2 (Mm00441242_m1), Ccl5 (Mm01302427_m1), Cxcl9 (Mm00434946_m1), Cxcl10 (Mm00445235_m1). custom primers for used for analyzing T. gondii genomic DNA and gene expression were used and are provided in S1 Table
1 mm zirconia silica beads
Manufactured by Biospec
Sourced in United States
The 1 mm zirconia/silica beads are a type of laboratory equipment designed for use in various applications. These beads are composed of a blend of zirconia and silica materials. They serve as an inert medium for physical processes such as mechanical disruption or mixing.
Automatically generated - may contain errors
Lab products found in correlation
Mini beadbeater, by Biospec (5 mentions)
Methylcellulose, by Merck Group (4 mentions)
Phenol red, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Mini beadbeater 16, by Biospec (1 mentions)
Custom primers, by Integrated DNA Technologies (1 mentions)
Taq based master mix, by Meridian Bioscience (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Tris edta buffer, by Avantor (1 mentions)
Puregene yeast bact kit, by Qiagen (1 mentions)
Fastprep bead beater, by MP Biomedicals (1 mentions)
12 protocols using 1 mm zirconia silica beads
1
Quantitative Molecular Profiling of Host and Parasite
2
Quantifying Brain mRNA Levels
3
Amplification of Protease-Resistant Prion Signals
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Serial dilutions of the seed were subjected to one-round of PMCA (24–48 h) and the proteinase-K (PK)-resistant signal obtained by Western blot was compared to the non-amplified seed. Reproducibility of the results was ensured by using 1 mm zirconia/silica beads (BioSpec Products)69 (link).
4
Quantifying Viral Plaque Assays in Lungs
5
Optimized Mycobacterium avium subsp. paratuberculosis Isolation
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The field isolate of MAP (strain 7072 from a white deer, RFLP type C1) was used throughout the whole study. The isolate was grown on Herrold’s egg yolk medium (HEYM) supplemented with 2 mg/ml Mycobactin J (Allied Monitor, USA) at 37 °C for 3 weeks until visible growth was observed. Afterwards, colonies were harvested and resuspended in 1.2 ml Tris–EDTA (TE) buffer (Amresco, USA) supplemented with Carrier DNA (salmon sperm DNA, final concentration 50 ng/µl; Serva, Germany). The MAP suspension was homogenized by vortexing for 30 s following the addition of 350 mg 1 mm zirconia/silica beads (Biospec, USA). In order to remove large MAP clumps, the suspension was centrifuged at 100 g for 30 s and the upper cell fraction was resuspended in TE buffer with Carrier DNA and diluted to an optical density at 600 nm (OD600) of 0.1, which corresponds to approximately 108 MAP cells/ml of suspension19 (link). A schematic overview of the entire experimental procedure is shown in Fig. 1 .![]()
Schematic overview of the entire experimental procedure.
6
Plaque Assay for Acute Phase Viral Titers
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Plaque assays were used to determine the tissue titers during the acute phase of infection. C57BL6/J mice were infected with MHV68.ORF73βla or MHV68.ZT6, and at the indicated time points, lungs were harvested from four mice per sample group per experiment. Plaque assays were then performed as previously described (87 (link), 88 (link)). Briefly, harvested lung tissues were placed in sterile 2-ml screw-cap tubes containing 1 ml of DMEM and 500 µl of 1-mm zirconia-silica beads (BioSpec Products) and stored at −80°C until use. Samples were thawed on ice, and tissues were homogenized using a mini-BeadBeater (BioSpec). Samples were serially 10-fold diluted in complete DMEM, added to single wells of a 6-well plate containing a monolayer of NIH 3T12 fibroblasts, and then overlaid with methylcellulose (Sigma). After 7 days, plaques were visualized by neutral red stain and counted.
7
Plaque Assay for Virus Quantification
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Plaque assays were performed as previously described (6 (link), 10 (link)). Briefly, harvested lungs were placed in sterile 2-ml screw-cap tubes containing 1 ml DMEM and 500 µl of 1-mm zirconia-silica beads (BioSpec Products) and stored at −80°C until use.
Samples were thawed on ice and then homogenized with a mini-beadbeater (BioSpec Products). Subsequently, samples were serially diluted 10-fold in serum-free DMEM, added to a single well of a 6-well plate containing a monolayer of NIH 3T12 fibroblasts, and then overlaid with a 1:1 mixture of methylcellulose (Sigma) and 2× Temin’s minimal essential medium modification, without phenol red (Invitrogen) and supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin. After 7 days, neutral red was used for plaque quantification.
Samples were thawed on ice and then homogenized with a mini-beadbeater (BioSpec Products). Subsequently, samples were serially diluted 10-fold in serum-free DMEM, added to a single well of a 6-well plate containing a monolayer of NIH 3T12 fibroblasts, and then overlaid with a 1:1 mixture of methylcellulose (Sigma) and 2× Temin’s minimal essential medium modification, without phenol red (Invitrogen) and supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin. After 7 days, neutral red was used for plaque quantification.
8
Viral Titer Quantification in Cells and Lungs
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To determine the titer of viral stocks, samples were prepared as 10-fold dilutions in serum-free DMEM, added to a single well of a six-well plate containing a monolayer of 2 × 105 NIH 3T12 fibroblasts, and then overlaid with 1:1 mixture of methylcellulose (Sigma, St Louis, MO, USA) and 2x MEM Temin’s modification, no phenol red medium supplemented with 5% fetal calf serum, 100 U/mL of penicillin, and 100 mg/mL streptomycin. After 7 days, neutral red stain was added to the assays to visualize and count plaques. To determine in vivo virus titers, lungs were harvested from four mice per sample group per experiment. Plaque assays were then performed as previously described [21 (link)]. Briefly, harvested lung tissues were placed in sterile 2 mL screw cap tubes containing 1 ml of DMEM and 500 μL of 1 mm zirconia-silica beads (BioSpec Products, Bartlesville, OK, USA) and stored at −80 °C until use. Samples were thawed on ice and tissues were homogenized using a Mini-BeadBeater (BioSpec Products). Samples were serially ten-fold diluted in complete DMEM, and plaque assays performed as described for virus stocks.
9
Murine Herpesvirus 68 Inoculation
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Eight- to 12-week-old mice were inoculated by the intranasal route with 1,000 PFU recombinant MHV68 in 20 μl of 10% DMEM or by intraperitoneal injection in 0.5 ml 10% DMEM under isoflurane anesthesia. The inoculum dose was validated by plaque assay titration. For acute replication, lungs were isolated and homogenized using 1-mm zirconia/silica beads (BioSpec, Bartlesville, OK) in a Mini-BeadBeater (BioSpec) and diluted for titration by plaque assay. For latency and reactivation assays, spleens were homogenized, treated to remove red blood cells, and passaged through a 100-μm-pore nylon filter. For PECs, 10 ml of 10% DMEM was injected into the peritoneal cavity, and approximately 8 ml of medium was withdrawn and centrifuged, and then the pellet was resuspended in 10% DMEM.
10
Virus Quantification in BV2 Cells
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