A retroviral overexpression system for CXCR4 was generated using the SF91 retroviral vector (Milsom et al., 2009 (link)). Viral supernatants were generated from Phoenix cells (ATCC). Cells were transfected with either SF-CXCR4 or SF-Venus using calcium phosphate transfection (Invitrogen). Retroviral supernatant was harvested 48 h after transfection, concentrated by ultracentrifugation, and stored at −80°C until use.
Calcium phosphate transfection
Manufactured by Thermo Fisher Scientific
Calcium phosphate transfection is a laboratory technique used to introduce genetic material, such as DNA, into cells. It involves the formation of a calcium phosphate-DNA co-precipitate, which is then added to the cells, facilitating the uptake of the genetic material.
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2 protocols using calcium phosphate transfection
1
Retroviral Overexpression of CXCR4
2
Poly(I:C) Stimulation of FLuDero Cells
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Establishment of the FluDero cell line has been described previously (Sarkis et al., 2018) (link).
The cell line was cultured in prewarmed DMEM/Ham's Nutrient Mixture F12 (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin and was maintained in a humidified incubator with 5% CO 2 at 37°C. For poly(I:C) stimulation, FLuDero cells were seeded at a density of 2.5×10 5 cells/well in 12-well plates. Cells were stimulated by transfection with 20 μg/mL of poly(I:C) (Sigma, cat #P9582) by calcium phosphate transfection (Invitrogen), 20 h after seeding. Cells were harvested in TRIzol Reagent at 0, 3, 6, 9, 12, 24, 48 and 72 h post-transfection and stored at -80°C until RNA extraction. The experiment was performed three times.
Total RNA was extracted from FLuDero cells transfected with poly(I:C) using TRIzol Reagent (Invitrogen) as recommended by the manufacturer. cDNAs were synthesized using Superscript ® III reverse transcriptase (Invitrogen) and random hexamers following the manufacturer's instructions.
The cell line was cultured in prewarmed DMEM/Ham's Nutrient Mixture F12 (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin and was maintained in a humidified incubator with 5% CO 2 at 37°C. For poly(I:C) stimulation, FLuDero cells were seeded at a density of 2.5×10 5 cells/well in 12-well plates. Cells were stimulated by transfection with 20 μg/mL of poly(I:C) (Sigma, cat #P9582) by calcium phosphate transfection (Invitrogen), 20 h after seeding. Cells were harvested in TRIzol Reagent at 0, 3, 6, 9, 12, 24, 48 and 72 h post-transfection and stored at -80°C until RNA extraction. The experiment was performed three times.
Total RNA was extracted from FLuDero cells transfected with poly(I:C) using TRIzol Reagent (Invitrogen) as recommended by the manufacturer. cDNAs were synthesized using Superscript ® III reverse transcriptase (Invitrogen) and random hexamers following the manufacturer's instructions.
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