TLR10 shRNA (h) Lentiviral Particles

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The human monocytic cell line THP-1 was obtained from ATCC (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% Antibiotic-Antimycotic, 10 mM HEPES buffer, and β-mercaptoethanol at 37 °C in a 5% CO2 humidified incubator.TLR10 knockdown THP-1 cell lines were established using stable expression of short hairpin RNAs (shRNAs) that target TLR10 mRNA. THP-1 cells were transduced with TLR10 shRNA lentiviral particles (sc-40272-V), control shRNA lentiviral particles (sc-108080), or copGFP control lentiviral particles (sc-108084) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).Transductions of THP-1 cells were carried out in the presence of 8 µg/mL of polybrene (sc-134220, Santa Cruz (Dallas, TX, USA)). After mixing with polybrene, the viral stocks were added to the cells (1 × 104 cells/well in 96 well plates) at multiplicity of infection (MOI) of 10. After 24 h of transduction, the cells were collected and then fresh media lacking polybrene were added to them. The transduced cells were allowed to proliferate until a sufficient cell number was reached for puromycin selection, which was performed in order to select stable clones expressing the shRNA. The cell culture medium was replaced with fresh medium plus 1 µg/mL of puromycin every 2 to 3 days until resistant clones appeared. After 3–4 weeks, the cells were collected and examined for GFP and TLR10 expression of the cells using FACS analysis. The selected clones were maintained in fresh puromycin-containing medium for an additional month, analyzed, and used for further experiments.

Protocol tips

The human monocytic cell line THP-1 was obtained from ATCC (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% Antibiotic-Antimycotic, 10 mM HEPES buffer, and β-mercaptoethanol at 37 °C in a 5% CO2 humidified incubator.TLR10 knockdown THP-1 cell lines were established using stable expression of short hairpin RNAs (shRNAs) that target TLR10 mRNA. THP-1 cells were transduced with TLR10 shRNA lentiviral particles (sc-40272-V), control shRNA lentiviral particles (sc-108080), or copGFP control lentiviral particles (sc-108084) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).Transductions of THP-1 cells were carried out in the presence of 8 µg/mL of polybrene (sc-134220, Santa Cruz (Dallas, TX, USA)). After mixing with polybrene, the viral stocks were added to the cells (1 × 104 cells/well in 96 well plates) at multiplicity of infection (MOI) of 10. After 24 h of transduction, the cells were collected and then fresh media lacking polybrene were added to them. The transduced cells were allowed to proliferate until a sufficient cell number was reached for puromycin selection, which was performed in order to select stable clones expressing the shRNA. The cell culture medium was replaced with fresh medium plus 1 µg/mL of puromycin every 2 to 3 days until resistant clones appeared. After 3–4 weeks, the cells were collected and examined for GFP and TLR10 expression of the cells using FACS analysis. The selected clones were maintained in fresh puromycin-containing medium for an additional month, analyzed, and used for further experiments.

Publication protocol

The human monocytic cell line THP-1 was obtained from ATCC (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% Antibiotic-Antimycotic, 10 mM HEPES buffer, and β-mercaptoethanol at 37 °C in a 5% CO2 humidified incubator.TLR10 knockdown THP-1 cell lines were established using stable expression of short hairpin RNAs (shRNAs) that target TLR10 mRNA. THP-1 cells were transduced with TLR10 shRNA lentiviral particles (sc-40272-V), control shRNA lentiviral particles (sc-108080), or copGFP control lentiviral particles (sc-108084) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).Transductions of THP-1 cells were carried out in the presence of 8 µg/mL of polybrene (sc-134220, Santa Cruz (Dallas, TX, USA)). After mixing with polybrene, the viral stocks were added to the cells (1 × 104 cells/well in 96 well plates) at multiplicity of infection (MOI) of 10. After 24 h of transduction, the cells were collected and then fresh media lacking polybrene were added to them. The transduced cells were allowed to proliferate until a sufficient cell number was reached for puromycin selection, which was performed in order to select stable clones expressing the shRNA. The cell culture medium was replaced with fresh medium plus 1 µg/mL of puromycin every 2 to 3 days until resistant clones appeared. After 3–4 weeks, the cells were collected and examined for GFP and TLR10 expression of the cells using FACS analysis. The selected clones were maintained in fresh puromycin-containing medium for an additional month, analyzed, and used for further experiments.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing shRNA gene silencing Human - THP-1 TLR10 using TLR10 shRNA (h) Lentiviral Particles from Santa Cruz Biotechnology.

Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for TLR10 shRNA (h) Lentiviral Particles below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing shRNA gene silencing Human - THP-1 TLR10 using TLR10 shRNA (h) Lentiviral Particles from Santa Cruz Biotechnology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.