Two populations of dCas9-TA cells are transduced with the CaT-A and CaT-B viral mix reagents, respectively. After 4 days, each population is analyzed by flow cytometry. The mean GFP and RFP fluorescent values are then used to calculate dCas9-TA activity in the dCas9-TA cells.

The assay was optimized using MDA-MB-231 cells. These dCas9-TA MDA-MB-231 cells are available from Cellecta to use in the assay as a positive control (see Additional Required Materials). Some protocol optimization may be needed based on the growth characteristics of your cells. If a negative control is also desired, the assay should be run with the parental cell line for the dCas9-TA cells.

Day 0

  1. Quickly thaw the CaT-A and CaT-B lentiviral particles in a water bath at 37°C. Transfer the thawed particles to a laminar flow hood, gently mix by rotation, inversion, or gentle vortexing, and keep on ice. Unused reagent can be aliquoted, refrozen at -80°C, and used again for subsequent experiments.
  1. Suspend dCas9-TA cells in growth medium supplemented with 1X Transduction Reagent, at a density of ca. 100,000 cells/ml.

Note: This cell density was calculated for MDA-MB-231 cells. Depending on cell size and growth, you may need to use a different concentration and correspondingly-sized plate. As a rule of thumb, cells should be transduced at a density such that they would become confluent in ~48 hours. For the assay, you should plate at least 100,000 cells.

  1. Aliquot 1 ml of cell suspension (100,000 cells) into each of 2 wells of a 12-well plate.
  1. Add 20 µl of CaT-A virus into one well and 20 µl of CaT-B virus into the other well, and then mix and return cells to incubator.

Note: For most cell lines, 20 µl of CaT viral reagents will suffice to obtain at least 30% RFP+ cells (the recommended minimum percentage of transduced cells for optimal assay sensitivity). For hard-to-transduce cell lines, more virus might be needed. If in doubt, it is recommended to set up two sets of transductions with 20 µl of CaT-A and CaT-B as described above, and 60 µl of each in the second transductions. For the final calculation, use the cell population which has closest to 50% RFP+ cells on Day 4. A control lentiviral vector may be used before running the assay to test the transduction efficiency of your cells (see Additional Required Materials).

Days 1-3

Exchange medium with fresh growth medium and grow cells under standard conditions for 3 days. Passage cells as needed. Cells should not become confluent.

Day 4

  1. Analyze cells by flow cytometry, using settings below:

Channel 1: excitation 488nM, emission 530/20nm (GFP)
Channel 2: excitation 561nM, emission 590/20nm (RFP)

  1. Use flow sorting software to calculate the mean GFP and mean RFP intensity for both CaT-A and CaT-B transduced cells, and then calculate the relative GFP/RFP intensity for each sample (normalized GFP intensity).

Last modified: May 26, 2017

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