The human PBX1 coding region and PARP1coding region was cloned into the pLVX-IRES-mCherry lentiviral vector (Youbio, China). The 10 μg lentiviral vector was cotransfected with 7.5 μg pMD2.G and 2.5 μg psPAX2 (Addgene) into 293T cells in a 100 mm cell culture plate using Lipofectamine (Invitrogen) 3,000 as transfection reagent. The viral particle were harvested at 48 and 72 h after transfection and concentrated by ultracentrifugation (Millipore, United States ). HF-MSCs were transduced with lentiviral particles encoding PBX1 or PARP1 or both PBX1 and PARP1 in the presence of polybrene (Santa Cruz, United States ) at final concentration of 10 μg/ml.
Ultracentrifugation
Manufactured by Merck Group
Sourced in United States, Germany
Ultracentrifugation is a laboratory technique that uses a high-speed centrifuge to separate and purify biological macromolecules based on their size, shape, and density. The process involves spinning samples at very high speeds, generating high gravitational forces that cause particles to sediment at different rates. This allows for the separation and isolation of molecules such as proteins, nucleic acids, and organelles.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Santa Cruz Biotechnology (2 mentions)
Plvx ires mcherry lentiviral vector, by YouBio (2 mentions)
Lipofectamine, by Thermo Fisher Scientific (2 mentions)
Pspax2, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pmd2 g, by Addgene (1 mentions)
Human cd8 t cell enrichment kit, by STEMCELL (1 mentions)
6 protocols using ultracentrifugation
1
Lentiviral Overexpression of PBX1 and PARP1 in HF-MSCs
2
Encapsulation Efficiency and Loading Efficiency Determination of Nanomaterials
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The encapsulation efficiency of NPs@FMN and MNPs@FMN was determined by ultrafiltration. Briefly, free FMN and NPs@FMN or MNPs@FMN were separated by ultracentrifugation (Millipore, Billerica, MA, USA). NPs@FMN or MNPs@FMN was retained, and free FMN was separated from the filtrated through the ultracentrifugation. The initial concentration of FMN in NPs@FMN or MNPs@FMN and free FMN were determined by UV–vis spectrometry. The encapsulation efficiency was calculated as follows:
where Wt refers to the initial concentration of the FMN to the NPs@FMN or MNPs@FMN, and Wf refers to the free FMN in the filtrate.
The collected samples of NPs@FMN and MNPs@FMN were lyophilized, and 1 mg freeze‐dried powder of NPs@FMN and MNPs@FMN were dissolved thoroughly in ddH2O and analyzed by UV–vis spectrometry. The loading efficiency was calculated as follows:
Where WF refers to the weight of the FMN in NPs@FMN or MNPs@FMN, and WN refers to the weight of NPs@FMN or MNPs@FMN.
where Wt refers to the initial concentration of the FMN to the NPs@FMN or MNPs@FMN, and Wf refers to the free FMN in the filtrate.
The collected samples of NPs@FMN and MNPs@FMN were lyophilized, and 1 mg freeze‐dried powder of NPs@FMN and MNPs@FMN were dissolved thoroughly in ddH2O and analyzed by UV–vis spectrometry. The loading efficiency was calculated as follows:
Where WF refers to the weight of the FMN in NPs@FMN or MNPs@FMN, and WN refers to the weight of NPs@FMN or MNPs@FMN.
3
Overexpression of PBX1 and PARP1 in HF-MSCs
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The overexpression of PBX1, PARP1, and PBX1 + PARP1 in HF-MSCs was achieved as previously described [15 (link), 20 (link), 21 ]. The human PBX1 coding region and PARP1coding region was cloned into the pLVX-IRES-mCherry lentiviral vector (Youbio, China). The 10 μg lentiviral vector was cotransfected with 7.5 μg pMD2.G and 2.5 μg psPAX2 (Addgene) into 293 T cells in a 100 mm cell culture plate using Lipofectamine (Invitrogen) 3,000 as transfection reagent. The viral particle were harvested at 48 and 72 h after transfection and concentrated by ultracentrifugation (Millipore, United States). HF-MSCs were transduced with lentiviral particles encoding PBX1 or PARP1 or both PBX1 and PARP1 in the presence of polybrene (Santa Cruz,United States) at final concentration of 10 μg/ml.
4
Isolation and Transduction of CD8+ T Cells
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Isolation of CD8+ T cells from the splenocyte suspension was performed using a human CD8+ T cell enrichment kit (STEMCELL, 19053, Canada), according to the manufacturer's protocol. The lentiviral supernatant containing the CAR construct was generated using the 293T packaging cell line, following established methods described in the literature. Briefly, 293T cells at 95 % confluency in 10-cm plates were co-transfected with 12 μg of CAR plasmids, 6 μg of pMD2.G, and 6 μg of psPAX2 packaging plasmid DNA using Lipofectamine 2000 (11668019, Thermo Fisher Scientific). The viral supernatant was collected at 48 and 72 h post-transfection, followed by ultracentrifugation (Millipore) at 4000 rpm for 1 h. The concentrated virus was subsequently stored at -80 °C for future use in experimental procedures.
5
Determining Liposomal Entrapment Efficiency
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The entrapment efficiency (E.E) of the CDDP@PLs was determined by the indirect method. Briefly, the liposomes were centrifuged by ultra-centrifugation (Sigma-Aldrich, Darmstadt, Germany) at 12,000 rpm for 40 min. The supernatant was collected for the quantification of the unentrapped drug and the process was repeated in triplicate for each sample. The drug was then estimated by taking absorbance through a UV/Visible spectrophotometer (IRMECO, 2020, Schwarzenbek, Lütjensee, Germany) [21 (link)]. The E.E was determined by the following formula:
6
Niosomal Drug Entrapment Efficiency
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The niosomal formulations were ultracentrifuged at 18,000 rpm for 30 min at 4 ℃ to determine DS entrapment. The ultracentrifugation (Sigma, Germany) was used to isolate the loaded DS from the dispersion. The amount of drug in the supernatant was then measured by ultravioletvisible (UV-vis) spectroscopy (UV-Vis Jasco V-630, UK) at a wavelength of the drug (DS) (276 nm). The entrapment efficiency percentage (EE%) was determined according to equation 1 [26] :
Where W initial is the quantity of total drug initially added to the formulations and W free is the quantity of the drug in the supernatant.
Where W initial is the quantity of total drug initially added to the formulations and W free is the quantity of the drug in the supernatant.
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