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Pes filter

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The PES filter is a type of laboratory equipment used for filtration processes. It is designed to remove particles, microorganisms, and other unwanted substances from liquids or gases, ensuring the purity and quality of the filtered material.

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Lab products found in correlation

Polybrene, by Thermo Fisher Scientific (3 mentions) Lenticrispr v2, by Addgene (3 mentions) Polybrene, by Merck Group (3 mentions) Packaging pcl eco plasmid, by Addgene (2 mentions) Lenticrispr v2, by New England Biolabs (1 mentions) Psecc, by Addgene (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Sodium pyruvate, by Merck Group (1 mentions) Pvsv g plasmid, by Takara Bio (1 mentions) Pcmv delta r8.2 plasmid, by Addgene (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Pcmv delta r8, by Addgene (1 mentions) Pcl eco, by Addgene (1 mentions) Hek293t 17 cells, by American Type Culture Collection (1 mentions) Polybrene, by Santa Cruz Biotechnology (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Mem non essential amino acids solution, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

19 protocols using pes filter

1

Generation of MTR Knockout Cells

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Generation of batch MTR knockout cells in each cell line was achieved using the lentiviral CRISPR–Cas9 vector system LentiCRISPR v2 (Addgene #52961). Briefly, RNA sequences targeting exon 15 and exon 28 of human MTR were designed using the crispr.mit.edu design tool. The guide RNA or scrambled control sequences (Supplementary Table 1) were subcloned into the LentiCRISPR v2 using the BsmBI restriction endonuclease (NEB R0580S). Virus was produced through PEI (MilliporeSigma, 408727) transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells. Medium containing lentivirus was collected after two days and filtered through a PES filter (0.22 μm, MilliporeSigma). Cells were transfected with virus containing scrambled control or targeting MTR and Polybrene (8 μg/mL, Invitrogen). Cells were split after 48 h into standard RPMI-media (10% FBS) containing puromycin (2 μg/mL) and cultured for 3 days. The resulting batch knockout cells were suspended in RPMI-media (20% FBS) and plated at a dilution of ~1 cell per well in 96-well plates. After 10 days, single colonies were identified and passaged. MTR knockout of clonally isolated knockout cells was confirmed by Sanger sequencing and by western blotting.
Ghergurovich J.M., Xu X., Wang J.Z., Yang L., Ryseck R.P., Wang L, & Rabinowitz J.D. (2021). Methionine synthase supports tumor tetrahydrofolate pools. Nature metabolism, 3(11), 1512-1520.
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2

CRISPR Knockout of G6PD in MDA-MB 231SCP:28TR Cells

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Generation of batch G6PD-null cells for mammary fat pad and tail-vein injection assays in the MDA-MB 231SCP:28TR background was achieved using the lentiviral CRISPR–Cas9 vector system lentiCRISPR v2 (Addgene #52961). Briefly, sgRNA sequences targeting exon-5 of human G6PD were designed using the crispr.mit.edu design tool. The identified PAM sequences or scrambled control sequence (Supplementary Table 1) were subcloned into the lentiCRISPR v2 using the BsmBI restriction endonuclease (NEB R0580S). Virus was produced through PEI (MilliporeSigma, 408727) transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells. Medium containing lentivirus was collected after two days and filtered through a PES filter (0.22 μm, MilliporeSigma). Cells were transfected with virus containing scrambled control or targeting G6PD and Polybrene (8 ug/mL, Invitrogen). Cells were split after 48 h into RPMI-media (10% FBS) containing puromycin (2 ug/mL) and cultured for 3 days. Cells were passaged once more in puromycin media prior to utilization in xenograft experiments. G6PD knockout was confirmed by western blotting.
Ghergurovich J.M., Esposito M., Chen Z., Wang J.Z., Bhatt V., Lan T., White E., Kang Y., Guo J.Y, & Rabinowitz J.D. (2020). Glucose-6-phosphate dehydrogenase is not essential for K-Ras-driven tumor growth or metastasis. Cancer research, 80(18), 3820-3829.
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3

Subcloning Murine G6pdx sgRNA Vectors

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sgRNA sequences targeting murine G6pdx and non-coding region of DNA (intergenic control) were identified from the mouse GeCKOv2 sgRNA library [36 (link)]. The identified PAM sequences (Supplementary Table 1) were subcloned into the pSECC (Addgene, 60820) using the BsmBI restriction endonuclease (NEB R0580S). Virus was produced through PEI (MilliporeSigma, 408727) transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells. Medium containing lentivirus was collected at 48 and 72 h and filtered through a PES filter (0.22 um, MilliporeSigma). Virus was concentrated by ultracentrifugation (~70,000g for 2 h at 4ºC), and the resulting pellet was gently resuspended in an appropriate volume of HBSS (MilliporeSigma, 55037C). Virus was aliquoted and snap-frozen in liquid nitrogen before storing at −80ºC. Viral titering was achieved using Green-Go cells and flow cytometry, as previously described [37 (link)].
Ghergurovich J.M., Esposito M., Chen Z., Wang J.Z., Bhatt V., Lan T., White E., Kang Y., Guo J.Y, & Rabinowitz J.D. (2020). Glucose-6-phosphate dehydrogenase is not essential for K-Ras-driven tumor growth or metastasis. Cancer research, 80(18), 3820-3829.
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4

Generation of MTR Knockout Cells

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Generation of batch MTR knockout cells in each cell line was achieved using the lentiviral CRISPR–Cas9 vector system LentiCRISPR v2 (Addgene #52961). Briefly, RNA sequences targeting exon 15 and exon 28 of human MTR were designed using the crispr.mit.edu design tool. The guide RNA or scrambled control sequences (Supplementary Table 1) were subcloned into the LentiCRISPR v2 using the BsmBI restriction endonuclease (NEB R0580S). Virus was produced through PEI (MilliporeSigma, 408727) transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells. Medium containing lentivirus was collected after two days and filtered through a PES filter (0.22 μm, MilliporeSigma). Cells were transfected with virus containing scrambled control or targeting MTR and Polybrene (8 μg/mL, Invitrogen). Cells were split after 48 h into standard RPMI-media (10% FBS) containing puromycin (2 μg/mL) and cultured for 3 days. The resulting batch knockout cells were suspended in RPMI-media (20% FBS) and plated at a dilution of ~1 cell per well in 96-well plates. After 10 days, single colonies were identified and passaged. MTR knockout of clonally isolated knockout cells was confirmed by Sanger sequencing and by western blotting.
Ghergurovich J.M., Xu X., Wang J.Z., Yang L., Ryseck R.P., Wang L, & Rabinowitz J.D. (2021). Methionine synthase supports tumor tetrahydrofolate pools. Nature metabolism, 3(11), 1512-1520.
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5

Staphylococcus aureus USA300 Metabolic Profiling

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Staphylococcus aureus strain ATCC BAA-1717 (USA300) were kindly provided by Abace Biotechnology (Beijing, China), and which had remarkable hemolytic activity and pigment formation. The bacteria were routinely cultured at 37°C on 5% sheep blood agar plates (BA, Bio-caring, China) and then grown in lysogeny broth (LB, Solarbio Life Sciences, Beijing, China) at 37°C with shaking at 180 rpm. Overnight cultures of S. aureus strain BAA-1717 were diluted 1:100 into 3 mL fresh LB medium with 20 mM glucose (Sigma-Aldrich) or/and 20 mM sodium pyruvate (Sigma-Aldrich) in a 12 mL tube. All cultures were incubated at 37°C with shaking at 180 rpm, and the culture supernatants were collected at 24 h or 48 h post-inoculation. The cell-free culture medium (CFCM) was obtained by filtered through a PES filter (0.22 μm pore size; Millipore), used immediately or stored at −70°C.
Chen T., Xu H., Yao X, & Luo Z. (2023). Role of sodium pyruvate in maintaining the survival and cytotoxicity of Staphylococcus aureus under high glucose conditions. Frontiers in Microbiology, 14, 1209358.
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6

Viral Transduction in HEK 293T Cells

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HEK 293T/17 cells (ATCC® CRL-11268, validated by ATCC) were used no longer than 4 weeks after thawing and were not tested for Mycoplasma. Retroviruses were prepared by co-transfecting HEK 293T/17 cells in a 10-cm plate with 10 μg of packaging pCL-ECO plasmid (Addgene #12371) and 10 μg MIGR1 based vectors by using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer’s protocol. Lentiviruses were generated by co-transfection of HEK 293T/17 cells with 10 μg of pLVX-CMV-hTet2(CD)-flag-IRES-mRFP1 based vectors, and 10 μg packaging pCMV delta R8.2 plasmid (Addgene #12263) and envelope pVSV-G plasmid (Clontech, #PT3343-5). Viruses were harvested 40h and 64h after the transfection and filtered through a 0.45-μm PES filter (Millipore). For infection, 5 × 105 cells were suspended in 1 ml of virus-containing medium with 6 μg/ml polybrene (Sigma). GFP+ and RFP+ cells were sorted 48 hrs after the initial infection.
Maifrede S., Le B.V., Nieborowska-Skorska M., Golovine K., Sullivan-Reed K., Dunuwille W.M., Nacson J., Hulse M., Keith K., Madzo J., Caruso L.B., Gazze Z., Lian Z., Padella A., Chitrala K.N., Bartholdy B.A., Matlawska-Wasowska K., Marcantonio D.D., Simonetti G., Greiner G., Sykes S.M., Valent P., Paietta E.M., Tallman M.S., Fernandez H.F., Litzow M.R., Minden M.D., Huang J., Martinelli G., Vassiliou G.S., Tempera I., Piwocka K., Johnson N., Challen G.A, & Skorski T. (2021). TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors. Cancer research, 81(19), 5089-5101.
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7

Retroviral and Lentiviral Transduction of Mouse Cells

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Ecotropic retroviruses were prepared by co-transfecting HEK 293 T/17 cells (ATCC® CRL-11268) in a 10-cm plate with 10 μg of packaging pCL-ECO (Addgene plasmid # 12371) and 10 μg retroviral based vectors by using Lipofectamine 2000 Transfection Reagent (Invirtogen) according to manufacturer’s protocol. For lentiviral infection, transfer vectors were co-transfected into HEK 293 T/17 cells with packaging pCMV delta R8.2 (Addgene plasmid # 12263) and envelope pVSV-G (Clontech, PT3343-5) vectors. Viruses were harvested 40 h and 64 h after the transfection and filtered through a 0.45-µm PES filter (Millipore). For viral infections, 5 × 105 mouse bone marrow cells or 32Dcl3 cells were resuspended in 1 ml of virus-containing medium with 6 μg/ml polybrene (Sigma). GFP, RFP or mCherryFP -positive cells were sorted 48 h after the initial infection.
Golovine K., Abalakov G., Lian Z., Chatla S., Karami A., Chitrala K.N., Madzo J., Nieborowska-Skorska M., Huang J, & Skorski T. (2023). ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias. Blood Cancer Journal, 13(1), 42.
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8

Labeling of sEVs with PKH26 and PbS QDs

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A total of 6.4 mg/mL HucMSC-derived sEVs were incubated with the lipophilic fluorescence dye PKH26 (MKBio, Shanghai, China) at a concentration of 10 μM at 37 °C. Then, the mixture solution was transferred to the ultrafiltration tube with 100 kDa MWCO and centrifuged at 1500× g for 30 min to remove the unbound dye. The stained sEVs were resuspended in 200 μL PBS and passed through a 0.22 μm polyethersulfone (PES) filter (Millipore, USA) before use. Similarly, 6.4 mg/mL HucMSC-, HEK293T-, and HGC-derived sEVs were incubated with 0.5 mg/mL of lead sulfide (PbS) quantum dots (PbS QDs) (Nirmidas Biotech, Mountain View, CA, USA) at 37 °C, and the mixture was processed in an ultrasonic water bath for 3 min. Then, the mixture was transferred to the ultrafiltration tube with 100 kDa MWCO to be centrifuged under 1500× g for 30 min to remove the free PbS QDs, and finally the obtained solution was added to 200 μL PBS with a 0.22 μm PES filter, ready for use. Control groups with only the label were performed, and the labeling efficiency was calculated by the ratio of fluorescence intensity before and after labeling.
Chen Y., Shi Y, & Tao Z. (2023). Fluorescence Tracking of Small Extracellular Vesicles In Vivo. Pharmaceutics, 15(9), 2297.
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9

Retroviral Transduction of Murine SMAD3 Constructs

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Dominant-negative murine SMAD3(D407E) mutant and SMAD3 wild-type (WT) cDNAs were obtained from Dr. Mitsuyasu Kato (Goto et al., 1998 (link)) and were re-cloned to pMIG-IRES-GFP retroviral construct. Retroviruses were prepared by co-transfecting HEK293T/17 cells (ATCC® CRL-11268) in a 10-cm plate with 10 μg of packaging pCL-ECO plasmid (Addgene #12371) and 10 μg MIGR1 based vectors by using Lipofectamine 2000 transfection reagent (Invirtogen) according to manufacturer’s protocol. Viruses were harvested 40h and 64h after the transfection and filtered through a 0.45-μm PES filter (Millipore). For infection, 5 × 105 cells were suspended in 1 mL of virus-containing medium containing 6 μg/ml polybrene (Sigma) and supplemented with recombinant growth factors, including 100 ng/mL SCF, 100 ng/mL FLT3, 20 ng/mL IL-3, and 20 ng/mL IL-6. GFP+ cells were obtained by fluorescent cell sorting 48 hr after the initial infection as described before (Dasgupta et al., 2016 (link)).
Le B.V., Podszywalow-Bartnicka P., Maifrede S., Sullivan-Reed K., Nieborowska-Skorska M., Golovine K., Yao J.C., Nejati R., Cai K.Q., Caruso L.B., Swatler J., Dabrowski M., Lian Z., Valent P., Paietta E.M., Levine R.L., Fernandez H.F., Tallman M.S., Litzow M.R., Huang J., Challen G.A., Link D., Tempera I., Wasik M.A., Piwocka K, & Skorski T. (2020). TGFβR-SMAD3 Signaling Induces Resistance to PARP Inhibitors in the Bone Marrow Microenvironment. Cell reports, 33(1), 108221.
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10

Lentiviral Transduction of Target Cells

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Lentivirus was generated in HEK-293T cells transfected with a transfer plasmid, psPAX2, and pMD2.G using polyethylenimine. Target cells were infected two days later with viral media filtered through a 0.45 μM PES filter (Millipore) with 8 μg/mL (final) polybrene (Santa Cruz). Cells were selected with 1-4 μg/mL puromycin.
Hosios A.M., Wilkinson M., McNamara M.C., Kalafut K.C., Torrence M.E., Asara J.M, & Manning B.D. (2022). mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species. Nature metabolism, 4(12), 1792-1811.
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