In general, OMVs were isolated following a procedure with minor adaptations20 (link). Bacteria were cultured overnight in RPMI 1640 (10-041-CV, Corning) with or without 10% FBS (10099141C, Thermofisher) at 30 °C, unless stated otherwise. The FBS was pre-cleared by ultracentrifugation (250,000 g, 4 °C,3 h) to remove native vesicles and filtered through a 0.22 μm filter (SLGPR33RB, Merck) before use. Bacterial cultures were centrifuged at 4,000 rpm for 20 min, the supernatants were sequentially filtered through a 0.45 μm filter (SLHP033RS, Merck) and a 0.22 μm filter (SLGPR33RB, Merck). The OMVs in the filtered supernatant were pelleted through subsequent ultracentrifugation (250,000 g, 4 °C,1 h) in 26 ml ultracentrifuge tubes (355654, Beckman) using a Backman 70Ti rotor. OMV pellets were washed in PBS and ultracentrifuge again. Washed OMV pellets were resuspend in 1.5 ml PBS (for quantification and biochemistry tests) or RPMI 1640 medium (for Plasmodium culture tests).
Slhp033rs
Manufactured by Merck Group
Sourced in United States
SLHP033RS is a laboratory equipment product manufactured by the Merck Group. It is designed to perform essential functions in a laboratory setting, though a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (6 mentions)
Fetal calf serum (fcs), by Merck Group (4 mentions)
Pcr4blunt topo vector, by Thermo Fisher Scientific (2 mentions)
T7 endonuclease, by New England Biolabs (2 mentions)
Pl crispr efs gfp, by Addgene (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Rpmi 1640, by Corning (1 mentions)
Slgpr33rb, by Merck Group (1 mentions)
Genejuice, by Merck Group (1 mentions)
Ls magnetic column, by Miltenyi Biotec (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Hatf00010, by Merck Group (1 mentions)
Vsv g, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Cas9 sgrna vector, by Addgene (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Polybrene tr 1003 g, by Merck Group (1 mentions)
Puromycin ant pr 1, by InvivoGen (1 mentions)
13 protocols using slhp033rs
1
Isolation of Outer Membrane Vesicles
2
Lentiviral vector production and transduction
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HEK293T cells were transfected with the plasmids encoding gag‐pol (psPAX2), VSV‐G(pMD2 G), and the plasmid containing the gene of interest (pRRL‐pUbC‐mNPHP3‐bPAC‐mCherry‐HA) using GeneJuice (Merck, #70967) according to the manufacturer’s instructions. The medium was exchanged to 30% FCS medium after 24 h. Two days post transfection, the supernatant was harvested and sterile‐filtered with a 0.45 μm filter (Merck, #SLHP033RS). Target cells were transduced by overnight exposure to a 1:2 mix of the harvested, filtered supernatant and growth medium, supplemented with 4 µg/ml of Polybrene (Sigma Aldrich, #H9268).
3
Lentiviral CRISPR-Mediated NKD2 Knockout
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The NKD2-specific guide RNA (forward 5′-CACCGACTCCAGTGCGATGTCTCGG -3′; reverse 5′-AAACCCGAGACATCGCACTGGAGTC -3′) were cloned into pL-CRISPR.EFS.GFP (Addgene #57818) using BsmBI restriction digestion. Lentiviral particles were produced by transient co transfection of HEK293T cells with lentiviral transfer plasmid, packaging plasmid psPAX2 (Addgene #12260) and VSVG packaging plasmid pMD2.G (Addgene #12259) using TransIT-LT (Mirus). Viral supernatants were collected 48-72 hours after transfection, clarified by centrifugation, supplemented with 10% FCS and Polybrene (Sigma-Aldrich, final concentration of 8μg/ml) and 0.45μm filtered (Millipore; SLHP033RS). Cell transduction was performed by incubating the PDGFRß cells with viral supernatants for 48hrs. eGFP expressing cells were single cell sorted into 96-well plates. Expanded colonies were assessed for mutations with mismatch detection assay: gDNA spanning the CRISPR target site was PCR amplified and analyzed by T7EI digest (T7 Endonuclease, NEB M0302S). To determine specific mutation events on both alleles within the clones grown, the PCR product was subcloned into the pCR™ 4Blunt-TOPO vector (Thermo Scientific K287520). Minimum 6 colonies per CRISPR-clone were grown and sent for sanger sequencing (Clone C2: 30 colonies have been sequenced).
4
Ookinete Microneme Secretion Protocol
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Ookinete microneme secretion was analyzed as previously reported (47 (link)). Briefly, about 2.0 × 106 of ookinetes were purified from in vitro culture using the LS magnetic column (#130-042-401, Miltenyi Biotec) and incubated in 500 μl of PBS for 4 hours at 22°C. The cells were spun down at 750g, and the supernatant was collected, filtered through a filter (0.45 μm; SLHP033RS, Millipore), and subjected to Western blot assay.
5
Preparing Viral DNA-Free Samples
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The culture medium of TW01-EBV cells after reactivation was harvested and passed through a 0.45-μm polyethersulfone (PES) membrane (Millipore, SLHP033RS). Filtered culture medium (50 μL) was treated with 2 U DNase I (Thermo Fisher, EN0521) per reaction at 37°C for 1 h, followed by 70°C heat inactivation for 30 min to remove virion-free DNA. The sample was then treated with 50 μg/mL protease K at 50°C for 1 h, followed by 75°C heat inactivation for 30 min.
6
Retroviral Transduction of NKD2 cDNA
7
Protein Fractionation and Analysis
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Proteins were prepared by homogenizing 4-w-old plant tissues in extraction buffer (20 mM Tris–HCl, pH 7.5, 200 mM NaCl, 10% glycerol, 60 μM MG132, 100 mM β- glycerophosphate, 20 mM sodium fluoride, protease inhibitors and PMSF) followed by 20 min of incubation at 4°C. After centrifugation at 13 000 rpm for 5 min, the supernatant was saved and filtered through a 0.45 μm filter (Millipore SLHP033RS). About 1.5 mg of total proteins were injected on the Superdex 200 10/300GL column (GE Healthcare Life Sciences GE17-5175-01) and fractionated by the AKTA fast protein liquid chromatography system (Amersham Biosciences). Proteins in each fraction were concentrated using acetone, separated by SDS-PAGE, and transferred onto nitrocellulose membranes (Millipore HATF00010) for immunoblot analysis.
8
Ookinete Microneme Protein Secretion
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Microneme secreted proteins were examined in in vitro ookinete culture supernatants as previously reported47 (link). 5.0 × 106 purified ookinetes were incubated in 200 μL PBS at 22 °C to allow microneme protein secretion. After 6 h incubation, the supernatants were collected by a centrifugation at 750 g for 3 min, filtered through 0.45 μm filter (Millipore, cat#SLHP033RS). Equal volume of 2 × Laemmli sample buffer was added. All samples were boiled at 95 °C for 10 min and centrifuged at 12,000 g for 5 min. Equal volume of supernatants from each parasite group were used for immunoblot analysis.
9
CRISPR-Cas9 Gene Editing of PAG1 in SH-SY5Y Cells
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CRISPR/Cas9 plasmids and sgRNAs targeting Exon 5 and 7 of the PAG1 gene were provided by Blake Wiedenheft and Royce Wilkinson (Montana State University). sgRNA sequences were housed in the lentiviral Cas9/sgRNA vector (Addgene #57828).
Exon 5 sgRNA: 5′-GAAGCCGCGACAGCATAGTG GGG-3′.
Exon 7 sgRNA: 5′-GCAGATCCGAGGCCGATGTC TGG-3′.
The plasmid carrying Cas9/sgRNA was cotransfected into HEK293FT cells with the lentiviral packaging vectors psPAX2 (Addgene #12260) and VSV-g (Addgene #8454) following the Lipofectamine 3000 protocol (ThermoFisher) to generate lentivirus. Supernatants containing lentivirus were filtered through a low-protein-binding syringe filter (0.45 μm, Millipore #SLHP033RS) before transductions. SH-SY5Y cells were transduced with different dilutions of virus-containing supernatant, and transfected cells were selected for by Puromycin (3 μg/ml) addition to culture media. Pools of puromycin-resistant cells were analyzed by Western blot for PAG1 expression, and those lacking PAG1 were characterized further. DNA surrounding the target site was amplified by PCR; PCR products were sequenced by Eurofins. While PAG1 expression appeared to be ablated in initial screens, we subsequently noted a truncated, soluble protein expressed in these cells (described in Results).
Exon 5 sgRNA: 5′-GAAGCCGCGACAGCATAGTG GGG-3′.
Exon 7 sgRNA: 5′-GCAGATCCGAGGCCGATGTC TGG-3′.
The plasmid carrying Cas9/sgRNA was cotransfected into HEK293FT cells with the lentiviral packaging vectors psPAX2 (Addgene #12260) and VSV-g (Addgene #8454) following the Lipofectamine 3000 protocol (ThermoFisher) to generate lentivirus. Supernatants containing lentivirus were filtered through a low-protein-binding syringe filter (0.45 μm, Millipore #SLHP033RS) before transductions. SH-SY5Y cells were transduced with different dilutions of virus-containing supernatant, and transfected cells were selected for by Puromycin (3 μg/ml) addition to culture media. Pools of puromycin-resistant cells were analyzed by Western blot for PAG1 expression, and those lacking PAG1 were characterized further. DNA surrounding the target site was amplified by PCR; PCR products were sequenced by Eurofins. While PAG1 expression appeared to be ablated in initial screens, we subsequently noted a truncated, soluble protein expressed in these cells (described in Results).
10
TMEM131 Knockdown in Osteosarcoma U2OS Cells
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Human TMEM131 knockdown in osteosarcoma U2OS cells was carried out with lentiviral shRNA (Sigma-Aldrich, SHCLNG-NM_015348). The TMEM131 shRNA targeting sequences are as follows: 5′-TAGCAGTTTCTCACCTATAAT-3′ [TRCN0000257459, CDS (coding sequence)], 5′-ATTATGCGCCAAGATCTAATT-3′ (TRCN0000246000, 3′UTR), 5′-TCCAATTGAGTTGGCTATAAA-3′ (TRCN0000246001, CDS), 5′-CTCGGACCCTTGGTCTAATTC-3′ (TRCN0000246002, CDS), 5′-CATAGATTGAGTGCTATATTT-3′ (TRCN0000246003, CDS). HEK293T was transfected by the pMD2.G, psPAX2, and shRNA plasmids, following the lentivirus production methods and manuals of TurboFect Transfection Reagent (Thermo Fisher Scientific, R0531). The lentivirus-based GFP-specific shRNAs were used as negative controls (Addgene, 31849). Forty-eight hours later, the TMEM131 shRNA lentivirus–containing media were collected and filtrated by a 0.45-μm syringe filter (Millipore EMD, SLHP033RS). The osteosarcoma U2OS cells were incubated with TMEM131 shRNA lentivirus medium for 24 hours in a humidified incubator at 37°C with 5% CO2. Transduction efficacy was enhanced by adding Polybrene (Sigma-Aldrich, TR-1003-G). Lentivirus-transduced cells were enriched by the medium with puromycin selection (1.5 μg/ml) for 3 days. Ascorbates were exogenously supplemented to ensure proper collagen modification. The knockdown efficiency of TMEM131 shRNA was evaluated by qRT-PCR.
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