How much polybrene do I add?

Add 4 µg/ml Polybrene and replace medium from target cells with the virus.

How do you add polybrene?

Infect cells with 2mls of the viral supernatant (or a dilution of the virus stock into 2mls) in the presence of 5ug to 10ug of polybrene per ml (final concentration). Incubate cells for 3 to 6 hours at 37°C. Add 8mls of complete medium. Three days after infection, split the cells 1:5 into selection medium.

How do you transduce a virus to a cell?

c. Transduction

Thaw the lentivirus on ice. Mix 8 µl Polybrene (1 mg/ml aliquot) with 957 µl culture.
The next day, exchange Lentivirus/Polybrene mixture by fresh culture medium. Incubate cells at standard cell culture conditions.
concentrations range from 0.1-10 μg/ml. Replace the culture medium 48-72 hours.

Is polybrene toxic to cells?

Polybrene is considered non-toxic at low concentrations, but has been found to negatively affect cell proliferation in some cell types, such as keratinocytes, at concentrations greater than 10 µg/mL [16].

How do you make a stable cell line protocol?

The protocol for generating stable cell lines requires several steps as shown below:

Generate a kill curve to determine the optimal selection antibiotic concentration.
Transfect cells with desired plasmid construct(s)
Select and expand stable polyclonal colonies.
Identify single clones by limited dilution and expansion.

How do you make polybrene stock solution?

A stock solution of polybrene can be prepared by dissolving 1 mg of polybrene in 1 mL sterile water with 10% DMSO; the stock solution is aliquoted and is stable at −20°C.

What is viral transfection technique?

Viral Transfection (Viral Transduction) This method involves the use of viral vectors to deliver nucleic acids into cells. Viral delivery systems such as lentiviral, adenoviral and oncoretroviral vectors can be used for transferring nucleic acids, even in hard-to-transfect cells.

How do lentiviruses work?

Lentiviruses (a genus of retrovirus) express reverse transcriptase, which converts the viral RNA to double stranded DNA, and integrase, which inserts this viral DNA into the host DNA. Once the viral DNA is integrated into the host DNA, it divides along with host cell and none are the wiser.

How do you store virus stock?

Keep the virus stock on ice. Sonicate twice for 30 sec in ice water, with a 30 sec rest on ice between, and divide it into 0.5- to 2-ml aliquots. Store the aliquots indefinitely at −80°C.

Do you need polybrene for transduction?

It is definitely possible to transduce your cells without the presence of polybrene or any other polycation.

How do you create a stable transfection?

Ensure that only one cell is present per well after the transfer.

Step 1 : Transfect cells. Transfect the cells using the desired transfection method.
Step 2 : Passage cells with antibiotic.
Step 3 : Monitor for cell “islands”
Step 4 : Isolate colonies.
Step 5 : Transfer single cells.

What should be the concentration of Polybrene in a well?

Since all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be 10 µg/mL. *Pro-Tip* Transducing too many cells relative to the number of virus particles reduces the transduction efficiency, resulting in massive cell death upon antibiotic selection.

When do colonies appear in general spinfection protocol?

This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed cells. It assumes that the four factor retrovirus being used has a high titer and that the BJ Fibroblasts being used are growing robustly. After transduction, colonies should start to appear within 3 to 5 weeks.

How to generate stable cell lines with lentivirus?

Procedure Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polyb 0 0 500 1:5 300 200 1:10 150 350 1:50 30 470

Can a lentiviral transgene be used for antibiotic selection?

Depending on the transducibility of the cell line used, this antibiotic selection may be a vital step for obtaining a population of cells that have taken up the lentiviral transgene. Note that not all lentiviral vectors deliver antibiotic resistance.

How much polybrene do I add? Add 4 µg/ml Polybrene and replace medium from target cells with the virus. How do you add polybrene? Infect cells with 2mls of the viral supernatant (or a dilution of the virus stock into 2mls) in the presence of 5ug to 10ug of polybrene per ml (final concentration). Incubate…