A colorimetric method to determine efficiency of FACS sorting

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The colorimetric method (Rodrigues et al, 2016) can be used to determine the efficiency of FACS sorting into 96- and 384-well plates. It’s an inexpensive method that uses 3, 3′, 5, 5′- Tetramethylbenzidine (TMB) and Horseradish Peroxidase (HRP) to verify whether a droplet from the cell sorter has successfully reached the fluid in the bottom of the well. The droplet contains the enzyme HRP, which is deposited into a well containing the substrate TMB.

When HRP and TMB come into contact, TMB is oxidised and rapidly turns a blue colour. This means that the user can quickly tell how many wells have been sorted correctly, and therefore how well the FACS sorter is calibrated immediately prior to a sorting experiment. This is particularly useful for single-cell experiments where the accuracy of the sorting is vital.

Required reagents

  • TMB (BioLegend PN 421501) – kept at 4°C, light sensitive
  • HRP (Life Technologies PN 31490) – kept at -20°C
  • 1x Phosphate buffered saline (PBS)


  1. Aliquot 2 µl TMB into all wells of a 96-well plate, or 1 µl TMB into a 384-well plate; depending on the plate type you will be using for the experiment.
  2. Prepare the cells (it is best to use the same cells that you will use for the experiment) as follows;
    1. Aliquot 500 µl cell suspension into a 1.5 ml tube – if your cells are adherent, you will need to lift them first.
    2. Spin the cells down at 500 x g for 4 minutes.
    3. Remove the supernatant without disturbing the cell pellet, and resuspend in 500 µl 1xPBS.
    4. Spin the cells down at 500 x g for 4 minutes.
    5. Remove the supernatant without disturbing the cell pellet, and resuspend in 375 µl 1xPBS.
    6. Add 125 µl HRP at a concentration of 250 µg/ml to the PBS-cell solution.
  3. Set up the FACS sorter, including all sorting alignment. Check the calibration by sorting 50 cells, from the HRP solution into every well of a plate with a lid on and make sure the droplets are in the centre of the wells. If not, make necessary alterations and re-test.
  4. Sort 1 cell into every well of the previously prepared plate with TMB.
  5. Immediately spin the plate down in a centrifuge.
  6. Check how many wells in the plate have tuned blue, this may take 5-10 minutes, and should be >90% for good sorting efficiency.

Note: if it’s not possible to use the same cells for both experiments, you can use FACS beads. However, it will give a less accurate representation of the sorting efficiency.

Examples of how the plates should look if the sort into a 96-well plate (A) or 384-well plate (B) was >90% successful, and if the sort efficiency is not good (~30%) (C)

Author: Laura Cubitt



Rodrigues OR, Monard S. A rapid method to verify single-cell deposition setup for cell sorters. Cytometry A. 2016 Jun;89(6):594-600. doi: 10.1002/cyto.a.22865. PubMed PMID: 27144818.