The stable transgenic cell lines for doxycycline-inducible expression of WT 52K, S28/75A, or S28/75D were generated by lentivirus transduction and puromycin selection of A549 lung adenocarcinoma cells. Lentivirus was generated by co-transfection of HEK-293T cells with lentivirus expression plasmid and two helper plasmids, pMD.G2 and pCMVΔR8.74 (see “Plasmids and transfections”), and the cells cultured in media containing serum that was heat inactivated at 56 °C for 30 min. Cell supernatant was collected at 48 and 72 h and filtered through 0.45-µM Acrodisc syringe filters (VWR, Cat#: 28143-312) using a 10-mL syringe. Cells were transduced by incubation with lentivirus supernatant in the presence of 10 µg/mL polybrene (Santa Cruz, Cat#: sc-134220) for 8 h. The supernatant was replaced with fresh lentivirus supernatant for an additional 16-h transduction. Lentivirus supernatant was then replaced with fresh culture media. Cells were cultured for a further 24 h before initial selection in 5 µg/mL puromycin (Gibco, cat#: A1113802). Cells were allowed to grow to confluency once under 5 µg/mL puromycin, before being shifted to a maintaining concentration of 1 µg/mL for all experiments.
Acrodisc syringe filter
Manufactured by Avantor
Sourced in Germany
Acrodisc syringe filters are a type of lab equipment used for the filtration of liquids. They are designed to remove particulates and contaminants from solutions prior to analysis or further processing.
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4 protocols using acrodisc syringe filter
1
Generating Stable Doxycycline-Inducible Cell Lines
2
Phage Enrichment from Fecal and Sewage
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Phage enrichment of processed fecal and sewage samples was conducted using previously established methods depicted in Figure 1 [24 (link)]. Filtrate (1 mL) was transferred into 5 mL of early-log-phase (OD600 = 0.2–0.3, ~107 CFU/mL) APEC culture grown in tryptic soy broth (TSB) (Sigma, Oakville, ON, Canada) containing 10 mM MgSO4 (mTSB). Filtrate/APEC cultures were then incubated at 37 °C in a shaking incubator at 150 rpm for 18–20 h. An extraction of 1.8 mL of culture was centrifuged at 22 °C, 11,000× g for 10 min and filtered using 0.2 µm Acrodisc syringe filters (Pall, VWR). Phages were enumerated using a soft-agar overlay plaque assay [25 ].
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CRISPR-Cas9 Mediated JCPyV Genome Editing
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Cas9 and gRNA was delivered into JCPyV permissive cells with high efficiency using a lentiviral vector (LentiCRISPR)47 (link) encoding Cas9, a gRNA and a puromycin selection marker (Fig. 1a ) was kindly provided by Dr. Feng Zhang.
The gRNAs target sequences in the JCPyV genome were designed using an online CRISPR design tool (http://www.genome-engineering.org/crispr/ ). Insertion of the gRNA targeting nucleotide sequence was performed as described47 (link). LentiCRISPR viruses were produced according to protocols accessible online at http://www.bu.edu/dbin/stemcells/protocols.php with minor modifications. Briefly, 12 μg of LentiCRISPR plasmid was co-transfected with 1.2 μg of VSV-g, 0.6 μg of gag/pol, 0.6 μg of rev and 0.6 μg of tat expression plasmids into 293FT cells in 10 cm dish using 45 μl of lipofectamine 2000 (11668027, Invitrogen). Supernatant containing the LentiCRISPR virus was collected 72 hours post transfection and centrifuged at 3000 rpm, at 4 °C for 10 min to remove cell debris. The supernatant was then filtered through 0.45 µm filter (28144–007, Acrodisc Syringe Filter, VWR) and aliquots were stored at −80 °C before use.
The gRNAs target sequences in the JCPyV genome were designed using an online CRISPR design tool (
4
Standardized Secretome Isolation Protocol
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Standard cell culture medium was conditioned by TM cells over 72 h. Depending on the proliferation rate, 3.75 × 106 fibroblasts, 2.25 × 106 JURKAT cells, 7.5 × 106 MIL4/IL13 macrophages, and 1.125 × 106 HUVEC cells were initially seeded in 15 cm cell culture dishes containing 17 mL medium. Afterward, conditioned medium (CM) was collected and pooled, sterile filtrated, and stored at −80 °C. Regarding harvesting of secretomes, GCT and TM cells were seeded in 15 cm cell culture dishes in standard cell culture medium. After 24 h, cells were washed five to seven times with 30 mL PBS before incubation in serum‐free medium for 24 h. Afterward, the medium was collected, centrifuged at 1000 g at 4 °C for 5 min, and filtered through a 0.2 μm Acrodisc syringe filter (VWR, Langenfeld, Germany) before being stored at −80 °C. As controls and for data normalization, the proteome of each cellular fraction was analyzed (each n = 3). Therefore, cells were harvested after being washed twice with 5 mL ice‐cold PBS. Cells were scraped into 1 mL of PBS, transferred into a 1.5‐mL tube, and centrifuged at 800 g at 4 °C. The supernatant was removed and the pellet stored at −80 °C. To evaluate the purity and quantity of the secretomes, a SDS/PAGE followed by a silver gel staining (Thermo Fisher Scientific, Schwerte, Germany) was performed after precipitation of 5 ml secretome by trichloroacetic acid.
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