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    Co-Culture Infection
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    Co-Culture Infection

    Introduction

    For the production of infected cell lines by Co-Culture Infection

    Materials

    • Cell Lines
      • Producer Cell Line: HEK293T (ATCC CRL-3216)
      • Target Cell Line: iSLK-Puro (Brulois et al. 2012)
    • Tissue Culture Material
      • DMEM + 10% FBS
      • Transfection Reagents
    • Induction Drugs
      • Sodium Butyrate (NaBut): 1 molar
      • 12-O-Tetradecanoylphorbol-13-acetate (TPA or PMA): 20 mg/mL
    • Selection Drugs
      • Hygromycin B: 50 mg/mL
      • Geneticin (G418): 50 mg/mL
      • Puromycin: 1 mg/mL
    • Purified BAC16
      • We use the Macherey-Nagal NucleoBond BAC 100 purification Kit

    Procedure

    • Day 1
    1. Plate 293T cells for transfection in a 6 well plate
    • If you are deleting an essential gene you will need to compliment the defect by making a stable 293T cell line with lentivirus first
    • Day 2
    1. Transfect the cells with your clean BAC prep using your transfection reagent of choice
    • We generally use PEI or PolyJet
    • Day 3
    1. Check to make sure that the transfection worked
    • The transfection isn't very efficient, given how large the BAC is, but there should be some GFP+ cells
    1. Trypsinize the transfected BAC16-293T cells as well as iSLK Puro (or other target) cells
    1. Count both both cell lines
    1. Mix the transfected BAC16-293T cells with the iSLK in a 1:1 ratio and plate in a 10cm dish
    • I generally use 1x106 of each cell type, but the exact number isn't critical
    • I use 10 mL of media for a 10 cm dish
    1. Allow the cells to settle down and attach for approximately 1 hour
    • You can also just leave them until the end of the day
    1. Add TPA (PMA) to 30nM and Sodium Butyrate to 300nM
    • Our stocks are 1000x TPA and 3000x NaBut for this application
    • Day 5
    1. The cells should be 100% confluent at this point, with the 293T cells forming a webbed pattern on top of the iSLK cells
    • See picture below for what they should look like
    1. Add an additional 5mL of media with TPA and NaBut to the dishes
    • This will ensure that the cells have enough nutrients while not removing any virus that is present
    • Don't add enough drugs for the whole 15mL, just enough for the new 5mL
    • Don't remove the supernatant or wash the cells, we want as much virus as possible in the dish
    • Day 7
    1. At this point, the 293T cells should be pretty green and the culture should look like the picture below

    Loading...

    • The "nodes" are 293T cells, which vary in percentage of GFP+ cells. This is a fairly normal looking culture at this point
    1. The induction media is removed and replaced with 10mL of fresh media containing the selection drugs, 300 μg/mL Hygromycin B, 1 μg/mL Puromycin, 250 μg/mL G418
    • Our stocks are: 166x Hygromycin B, 1000x Puromycin, 200x G418
    • The Hygromycin B will kill uninfected cells, and the Puromycin and G418 will kill all 293T cells
    • Day 7 - 14
    1. At this point, the cells should be checked every other day for cell death
    1. The media should be changed to fresh media with selection drugs every other day (or 3x a week)
    • You can also trysinize the cells and let them sit back down to speed things up, but I generally don't do this
    1. The cells should die at a pretty steady pace, starting with the 293T cells. Almost all of the 293T cells will be gone by the time the iSLK cells start to die.
    • Once the 293T cells are gone, you should be able to see GFP+ iSLK cells, but they will be a much fainter green than the BAC16-293T cells were
    1. The Hygromycin B concentration should be increased once you stop observing cell death, I increase the concentration according to the series below.
    • 300 μg/mL > 500 μg/mL > 700 μg/mL > 1 mg/mL (maintenance concentration)
    1. As the cells grow to fill the 10cm dish they should be moved to a 15cm plate. They can then be split as normal.
    1. I freeze ~5 vials of cells as soon as they are acting normally and then characterize as soon as I can.
    • For normal passaging, I only use 1 μg/mL Puromycin and 1 mg/mL Hygromycin

    Loading...

    • This is what my cells looked like 7 days after adding selection media. You can see that the cells are no longer 100% confluent, the 293T cells are contacting and balling up, and the GFP signal has spread from the 293T "nodes" to the iSLK cells in the top-middle and top-right.

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