The concentration of transfection units per milliliter (TU/mL) was calculated using the following formula: (% infected cells × cells used in the titration/100 × 1000 μl/μl virus added to the well) = TU/mL. The stable overexpression and inhibition of miR-140-5p in P3-DPCs were established by stable transduction with lentivirus (1E + 8 TU/mL) (Sino Biological) at a multiplicity of infection (MOI) of 10. In brief, DPCs (P3) were transfected with miRNA oligonucleotides containing an 50 nM miR-140-5p overexpression sequence and the corresponding negative control vector (miRNA-NC), or an 50 nM miR-140-5p inhibiting segment (si-miR-140-5p) and the corresponding negative control vector (si-miRNA-NC), all purchased from Sino Biological. In both the SiHa and C33a cell lines, the transfected cells were designated as miRNA-NC, miR-140-5p, si-miRNA-NC, and si-miR-140-5p, and DPCs treated with an equal volume of PBS were used as a blank control. After 12 h of culture, EVs (miRNA-NC-EV, miR-140-5p-EV, si-miRNA-NC-EV, and si-miR-140-5p-EV) were isolated from DPCs and used for subsequent experiments. An equal amount of PBS was used as a blank control.
Lentivirus
Manufactured by Sino Biological
Lentivirus is a type of replication-deficient viral vector derived from the lentivirus family. It is commonly used as a tool in molecular biology and biotechnology research for gene delivery and expression. Lentivirus can efficiently transduce both dividing and non-dividing cells, and is capable of integrating the target gene into the host cell genome, enabling long-term gene expression.
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3 protocols using lentivirus
1
Modulation of miR-140-5p in Dermal Papilla Cells
2
SARS-CoV-2 Spike Protein Lentivirus Mimic
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Spike S1 (40591-V08H; Sino Biological) was conjugated to lentivirus (Cellomics Technology LLC) to create a SARS-CoV-2 mimic. His-tagged spike S1 was linked to Ni nitrilotriacetate (Ni-NTA) through the chemical interaction. NTA with mercapto group (N-[Nα,Nα-Bis(carboxymethyl)-L-lysine]-12-mercaptododecanamide) was first reacted with 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) to give NTA-SMCC and then was added to the lentivirus. The NTA groups were conjugated to the lentivirus through the −NH2 group on lentivirus and N-hydroxysuccinimide ester on NTA-SMCC. The free NTA-SMCC was removed by centrifugation using an ultrafiltration tube (100 kDa MWCO; Millipore) to give SARS-CoV-2 mimicking virus (spike S1-lentivirus). The successfully conjugated spike S1 on lentivirus was confirmed using TEM. Briefly, SARS-CoV-2 mimics were incubated with anti-Spike S1 antibodies overnight at 4°C. Free antibodies were removed using an ultrafiltration tube (100 kDa MWCO; Millipore) and washed with PBS three times. Spike S1 on the SARS-CoV-2 mimics was labeled with immunogold (10 nm) antibodies and negatively stained for TEM visualization. The conjugation efficiency of spike S1 on lentivirus was determined using ELISA (Sino Biological SARS-COV-2 SPIKE ELISA KIT, Sino Biological) according to manufacturer’s protocol.
3
SARS-CoV-2 Spike Protein Lentivirus Mimic
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