Conditioned media was prepared as described previously [7 (link)]. Briefly, cells were allowed to reach 80–90% confluence, and counted to ensure proper multiplicity of infection (MOI). Cells were infected with or without HSV1716 or UV-inactivated HSV1716 and incubated at 37 °C 5% CO2 for 24 h. The conditioned media was harvested under sterile conditions and centrifuged at 4000× g for 10 min. Then, supernatants were filtered once using a 0.22 µm Steriflip filter (EMD Millipore, Darmstadt, Germany) followed by a final filtration using a 0.1 µm filter (Sartorius AG, Goettingen, Germany). Conditioned media from virus infected cell lines underwent plaque forming assay to ensure complete virus removal.
Steriflip filter
Manufactured by Merck Group
Sourced in United States, Germany
The Steriflip filter is a laboratory filtration device designed to facilitate the sterile filtration of liquids. It operates by utilizing a hydrophilic PVDF membrane with a pore size of 0.22 microns to remove particulates and microorganisms from samples.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (5 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
0.1 m filter, by Sartorius (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
T25 flask, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
48 well plate, by Greiner (1 mentions)
Bacteriological peptone, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Avantor (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Lyophilization, by Labconco (1 mentions)
10 dg desalting column, by Bio-Rad (1 mentions)
Protino, by Macherey-Nagel (1 mentions)
Sigmafast protease inhibitor, by Merck Group (1 mentions)
Rna bacteria protect reagent, by Qiagen (1 mentions)
28 protocols using steriflip filter
1
Preparation of Conditioned Media for Virus Studies
2
SARS-CoV-2 Isolation and Viral Titration
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Material from the original clinical specimens (NP swabs in UTM) were mixed 1:1 with high glucose DMEM (Gibco Thermo Fisher Scientific) supplemented with 5% FBS (Cellgro Mediatech), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 100 µg/mL gentamycin (Thermo Fisher Scientific). The mixture was passed through a 0.45 µm steriflip filter (Merck Millipore, Burlington, MA, USA) and used to inoculate Vero cells (1 × 106 cells/well) in a 48-well plate (Greiner Bio-One, Kremsmünster, Austria). Cells were monitored daily for cytopathic effect (CPE) for five days, and cell-free supernatant from positive cell cultures was used to inoculate 3 × 106 Vero cells in a T-25 flask (Thermo Fisher Scientific). These initial viral stocks (first serial passage or C1) were titrated by determining tissue culture dose for 50% infectivity (TCID50) in triplicate with CPE as the end-point using the Reed and Muench method [28 ]. SARS-CoV-2 titers were expressed as TCID50 per milliliter (TCID50/mL).
3
Bacterial Composition Analysis of Fermented Meats
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From each fermented meat product, two samples were taken, representing biological duplicates, to analyze the bacterial composition. Bacterial DNA was extracted by first centrifuging a homogenized mixture of sample and recovery diluent at 900× g for 10 min to remove coarse impurities. This recovery diluent consisted of a sterile solution of 0.85% (m/v) NaCl (VWR International, Darmstadt, Germany) and 0.1% (m/v) bacteriological peptone (Oxoid, Basingstoke, Hampshire, UK). Thereafter, the resulting suspensions were filtered through a 20-μm average pore-size 50-mL Steriflip filter (Merck, Darmstadt, Germany) to remove remaining contaminants. Cell pellets were then obtained by centrifuging at 4000× g at 4 °C for 20 min.
Thereafter, DNA extraction was carried out, using a combination of enzymatic, chemical, and mechanical cell lysis. This was followed by phenol/chloroform/isoamyl alcohol extraction and column purification of the DNA, as described in [34 (link)], except that the enzymatic steps using lyticase and Zymolyase were excluded from the protocol as molds or yeasts were not targeted in this study.
Thereafter, DNA extraction was carried out, using a combination of enzymatic, chemical, and mechanical cell lysis. This was followed by phenol/chloroform/isoamyl alcohol extraction and column purification of the DNA, as described in [34 (link)], except that the enzymatic steps using lyticase and Zymolyase were excluded from the protocol as molds or yeasts were not targeted in this study.
4
CRISPR/Cas9 Knockout Cell Line Generation
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The CRISPR/Cas9 and mouse sgRNA-RFP plasmids were obtained from Addgene (Plasmid #52961) and Sigma, respectively. The guide empty vector was used to serve as the control (Sigma). Twenty microliters of X-tremeGENE9 DNA transection Reagent (Roche) were mixed with 500 μl Opti-MEM medium (Gibco) for 5 min, incubated with 6 ng of DNA (CRISPR/Cas9, 090, 091 plasmids) for 25 min at room temperature, and these mixtures were added to HEK293T cells for 24 h. The medium with the viruses was harvested and filtrated through a 0.45 µm Steri-Flip filter (Millipore). The collected medium was mixed with 20% fresh culture medium and added to 4T1 cells along with 7 μg/ml polybrene for 4 h and replaced with normal 10% fresh medium. Two days after infection, 4T1 cells were selected with 4 μg/ml blasticidin or 5 μg/ml puromycin for 5 days. Multiple clones derived from single cell cultured in 96-well plates were isolated. Confluent cells were then plated in a 24-well plate and knockout cell clones were selected by sequencing. The sequencing data from knockout cells were shown in Supplementary Table S1 .
5
Lentiviral Transduction and Selection
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Expression constructs were transfected into cancer cell lines using Lipofectamine LTX reagent (Life Technologies) by following the manufacturer’s protocol. Lentivirus was produced by co-transfecting 293T cells with pRSV-Rev, pMD2.G, pMDLg/pRRE, and pLKO.1-puro shRNA or pCDH-puro expression vectors. Virus was harvested after 48 hours by filtering the virus-containing medium through 0.45 µM Steriflip filter (Millipore). Virus infection was performed by incubating cells with medium containing indicated virus and 8 µg/mL polybrene (Sigma) for 24 hours. Cells were allowed to recover in complete medium for 24 hours and then selected with puromycin for 24 hours (mRNA analysis) or 48 hours (protein and phenotypic analyses). Surviving pools were subjected to indicated experiments. Note that HK-2 cells undergoing virus infection and puromycin selection typically need 10–14 days recovery time before subjected to experiments.
6
Isolation and Characterization of Exosomes
7
Purification of Histidine-Tagged Protein
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The pellet was transferred
to PBS buffer and centrifuged for 10 min at 4300 rcf. The resulting
pellet was lysed in 30 mL of lysis buffer, referred to hereafter as
buffer B (20 mM bicine, pH 8.5, 500 mM NaCl, 10 mM imidazole), supplemented
with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma-Aldrich),
5 mM PMSF, and 2 mg of lysozyme. Without incubation, the resuspension
was lysed with an Avestin C3 homogenizer followed by a 20 min centrifugation
at 24,000 rcf at 4 °C. The supernatant was filtered through a
40 μm Steriflip filter (Millipore) and loaded onto a 5 mL NiNTA
column (Protino, Machery Nagel) connected to an Akta purifier preequilibrated
with buffer B. The column was washed with 50 mL (10 column volumes)
of 20 mM bicine (pH 8.5), 500 mM NaCl, 10 mM imidazole, 10 mM β-ME.
The protein was eluted with 20 mM bicine (pH 8.5), 500 mM NaCl, 250
mM imidazole, 10 mM β-Me. Imidazole was removed by exchanging
against 20 mM bicine (pH 8.5), 500 mM NaCl, with a 10DG desalting
column (BioRad). For purposes of lyophilization, the protein was directly
exchanged against 20 mM bicine (pH 8.5) and 100 mM trehalose, followed
by flash freezing with liquid N2 and lyophilization (Labconco)
overnight. Typical protein yields are 800 μM (3 mL total) from
a 1 L culture, with a purity of ∼98% by SDS–PAGE and
LC–MS (ESI-TOF) (6224 TOF and 1200 series HPLC, Agilent Technologies).
to PBS buffer and centrifuged for 10 min at 4300 rcf. The resulting
pellet was lysed in 30 mL of lysis buffer, referred to hereafter as
buffer B (20 mM bicine, pH 8.5, 500 mM NaCl, 10 mM imidazole), supplemented
with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma-Aldrich),
5 mM PMSF, and 2 mg of lysozyme. Without incubation, the resuspension
was lysed with an Avestin C3 homogenizer followed by a 20 min centrifugation
at 24,000 rcf at 4 °C. The supernatant was filtered through a
40 μm Steriflip filter (Millipore) and loaded onto a 5 mL NiNTA
column (Protino, Machery Nagel) connected to an Akta purifier preequilibrated
with buffer B. The column was washed with 50 mL (10 column volumes)
of 20 mM bicine (pH 8.5), 500 mM NaCl, 10 mM imidazole, 10 mM β-ME.
The protein was eluted with 20 mM bicine (pH 8.5), 500 mM NaCl, 250
mM imidazole, 10 mM β-Me. Imidazole was removed by exchanging
against 20 mM bicine (pH 8.5), 500 mM NaCl, with a 10DG desalting
column (BioRad). For purposes of lyophilization, the protein was directly
exchanged against 20 mM bicine (pH 8.5) and 100 mM trehalose, followed
by flash freezing with liquid N2 and lyophilization (Labconco)
overnight. Typical protein yields are 800 μM (3 mL total) from
a 1 L culture, with a purity of ∼98% by SDS–PAGE and
LC–MS (ESI-TOF) (6224 TOF and 1200 series HPLC, Agilent Technologies).
8
Lentiviral Transduction and Selection
9
Bacterial Coculture Transcriptome Analysis
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Cell samples from bacterial co-cultures with tomato roots were extracted as follows: the suspension was filtered with a 20 μm Steriflip filter (Millipore Corp.) to eliminate all plant debris. Filtrate was centrifuged at 8000 × g and pellet was collected and suspended in RNA Bacteria Protect Reagent (Qiagen Cat. 76506) following the manufacturer’s instructions. Prepared samples were stored at −70°C for further processing. Total RNA was extracted from the stored samples as per Jahn et al. [57 (link)]. Quantity of extracted RNA was measured using the Nanodrop 2000C spectrophotometer. Quality was assessed on denaturing electrophoresis gels. Purified RNA was reverse-transcribed to cDNA using the SuperScript® ViloTM cDNA Synthesis Kit (LifeTechnologies Cat. 11754–050), according to manufacturer’s instructions. Quality and quantity of cDNA were assessed by spectrophotometry (Nanodrop Technologies, Inc.).
10
Cigarette Smoke Extract Preparation
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Cigarette smoke extract (CSE) was prepared from research-grade cigarettes (3R4F) purchased from the Kentucky Tobacco Research and Development Center (University of Kentucky, Lexington, KY). 100% CSE was generated by bubbling two cigarettes into 20 mL of PBS (pH 7.4, without calcium and magnesium) at a rate of 1 cigarette/min to 0.5 cm above the filter followed by readjusting the pH to 7.4 and filtration using a 0.22 µm Steri-flip filter (Millipore, Bedford, MA). Air control extract was prepared using a similar procedure by replacing the cigarette with ambient air bubbled into PBS. Endothelial cells were treated with 2.5% CSE diluted in serum-free DMEM.
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