Jan 12, 2026
Staining for FACS Sorting of Live Cells
- Nahum Smith1,
- Silvia Casadei1
- 1University of Washington
Protocol Citation: Nahum Smith, Silvia Casadei 2026. Staining for FACS Sorting of Live Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnjozvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 07, 2026
Last Modified: January 12, 2026
Protocol Integer ID: 238186
Keywords: saturation genome editing experiment, live cell, live cells this protocol, cell, fac
Abstract
This protocol describes Hoechst 33342 staining and FACS sorting of Hap-1 cells in preparation for Saturation Genome Editing experiments.
Materials
**Required reagents**
Hoechst 33342, 12.3 mg/ml in aqueous solution (20mM); ThermoScientific # 62249
PBS, 1x
IMDM+10%FBS
FBS (heat inactivated, 1mL)
Trypsin
Trypan blue
**Required plasticware**
Cell counter
50mL Falcon tubes, 2 sets, for collection and preparation of conditioned media
50mL Falcon tube for harvested cells
15mL Falcon tubes, 2 sets, to carry cells before and after sorting
50mL conical tube with filter
Cell strainer
Troubleshooting
Before start
Prepare a 1mg/mL dilution of Hoechst 33342 (12.3 mg/ml) in water. Determine the amount of dye needed based on the number of culture dishes that need staining and harvesting. Hoechst 33342 precipitates in PBS; only use ddH2O for dilutions. Keep dye protected from light.
Recommended Hoechst 33342 (HO) staining concentration for live cells is 1-5ug/mL. To determine the optimal concentration of Hoechst 33342 for live staining of HAP1 cells, we ran a calibration experiment using 2, 4, 8ug/mL live staining and obtained optimal results with 2ug/mL as well as 4ug/mL Hoechst 33342. We chose to use 2ug/mL Hoechst 33342 for a sorting experiment.
Preparing Staining Media
1h
To perform staining in vitro, both media and trypsin should contain the same HO concentration. Prepare enough IMDM+10%FBS+0.2%HO and Trypsin+0.2%HO for the number of culture plates that need staining.
For each 15cm plate, prepare the following:
- 30mL IMDM+10%FBS+0.2%HO (20mL for staining incubation at 37C, 10mL to neutralize trypsin)
- 2mL Trypsin+0.2%HO
- 20mL PBS+1%FBS (assuming 100x10^6 cells in a 15mL plate and resuspending cells at 5x10^6 cells/mL for sorting)
**Prepare 50% conditioned media. At least 01:00:00 before the scheduled sorting time, label 50mL Falcon tubes for conditioned media. Transfer culture media (instead of aspirating) from cell plates to a 50mL Falcon tube, centrifuge at 1000rpm for 5-10minutes, collect supernatant and filter in a fresh 50mL tube. Add same volume of fresh IMDM+10%FBS to obtain 50% conditioned media.
1h
Staining and harvesting cells
45m
Collect and transfer media from culture plate to a 50mL Falcon tube and prepare conditioned media.
Wash culture plate with 10mL PBS once.
Add 20mL IMDM+10%FBS+0.2%HO media and incubate at 37°C for 00:45:00 (live staining).
Set up trypan blue and cell counter.
45m
Aspirate media and wash once with PBS. Add 2mL Trypsin + 0.2%HO and incubate at 37°C for 4 minutes. Make sure cells are detaching from plate by hitting gently on side of hood. Neutralize trypsin by adding 10mL IMDM+10%FBS+0.2%HO to the cells, gently resuspend and collect cells in a 50mL Falcon tube.
Resuspend cells homogeneously. Count cells and determine resuspension volume to a concentration of 5x10^6 cell/mL.
Centrifuge cells at 500rpm for 5-10 minutes. Discard supernatant and resuspend in PBS+1% heat-inactivated FBS (Or equivalent sorting buffer) to 5x10^6 cell/mL.
If sorting 100x10^6 cells, resuspend cell pellet in 20mL sort buffer. Using a cell strainer to break cell clumps, aliquot 5mL cells each in 15mL Falcon tubes.
Label 15mL Falcon tubes for collection of sorted cells. Add 1mL conditioned media to each tube and seal. Keep sterile.
Prepare a clean storage box to transport cells to FACS machine. Bring clean and labelled 15mL Falcon tubes (with 1mL conditioned media) for cell collection, gloves, notebook.
FACS
2w
Calibrate FSC and SSC according to your machine's settings.
Establishing gating with an unstained sample is recommended.
Most FACS machines DAPI laser is sufficient for Hoechst 33342 stain.
Final gate should be drawn around 1n peak to sort for halpoid cells. A well-sorted cell population will have no 4n peak.
FACS plot depicting 1n, 2n, and little to no 4n peak
Sort into conditioned media before transferring to 10cm plate for outgrowth. Expect relatively large volumes of sort buffer in output tube, so gentle homogenization and supplementation of extra media in outgrowth plates is recommended.
It is not recommended to pellet and resuspend recently sorted cells.
Replace growth media with fresh growth media 24hr after sorting.
Verification
After sufficient outgrowth (Typically 50-100 vials of 10 million cells per vial), perform a ploidy check on a population of grown cells.
We recommend checking cells that have been frozen, thawed, and allowed 1 passage before verifying ploidy, as this mimics the pre-transfection conditions for Saturation Genome Editing experiments.
2w