Jul 02, 2026

Fluorescence-activated cell sorting (FACS)

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwx987gmk/v1
  • Bruno Pavletić1,
  • Mario Stojanović1,
  • Ana Runtić1,
  • Roberto Galov1,
  • Dragomira Majhen1
  • 1Ruđer Bošković Institute
  • AdenoTeam
Bruno Pavletić
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwx987gmk/v1
Protocol Citation: Bruno Pavletić, Mario Stojanović, Ana Runtić, Roberto Galov, Dragomira Majhen 2026. Fluorescence-activated cell sorting (FACS). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwx987gmk/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: In development
We are still developing and optimizing this protocol
Created: June 18, 2026
Last Modified: July 02, 2026
Protocol  Integer ID: 319386
Abstract
Purpose and Scope
This protocol outlines a optimized workflow for the preparation, fluorescent immunostaining, and analysis of single-cell suspensions using flow cytometry and fluorescence-activated cell sorting (FACS). Developed at the Ruđer Bošković Institute, cell signal transduction group. This method is specifically designed for evaluating cell surface receptors on cultured cells.
Methodology Cell Harvesting and Quantification: Cells are harvested from 10 cm Petri dishes, washed sequentially, and resuspended. Cell density is determined using a specialized cell counting chamber slide under a microscope using a precise zig-zag counting pattern. Immunostaining: Cells are sequentially labeled with a primary antibody (1:99 dilution) and a fluorescently-conjugated secondary antibody (1:50 dilution). Both incubation steps last for 1 hour on ice with periodic manual agitation. Flow Cytometry Run: Stained cell suspensions are transferred to specialized FACS tubes and evaluated using a flow cytometer, such as the BD FACSymphony A1. The workflow incorporates an untreated control sample to calibrate the optical filters based on the designated fluorescent dyes.

Critical Parameters Temperature Control: A defining element of this protocol is the mandatory use of ice-cold PBS for all resuspension and wash steps following cell counting. Maintaining a low temperature effectively halts endocytosis, preventing the internalization of antibodies and ensuring accurate targeting of surface-bound receptors. Agitation: Mechanical agitation via vortexing is restricted to initial cell preparation steps ; gentle finger-tapping must be used during antibody incubations and washes to maintain cell integrity.
Guidelines
Cell Harvesting & Pipetting

  • Pipette Selection: Use 5 mL glass pipettes with a pipette boy when washing cells with 5 mL of PBS.
  • Dissociation Wash: Use 10 mL glass pipettes with a pipette boy when washing cells with 2 mL of versene, trypsin, or PBS, and during the subsequent 8 mL PBS resuspension.
  • Mixing: Perform all early washing steps at room temperature and mix gently.


Cell Counting

  • Counting Pattern: Count your cells on a plate counter slide under a microscope across five large squares using a "zig-zag" pattern, starting from any top square.
  • Boundary Rules: Adhere to the strict boundary rule: count cells touching the top or right borders, but do not count cells touching the bottom or left borders.
  • Chamber Maintenance: After utilizing a chamber on the plate counter slide, physically write an "x" on it to mark it as used.

Antibody Incubation & Handling

  • Incubation Aggregation: Keep the cells on ice for 1 hour for both primary and secondary antibody steps. Gently mix the solution every 15 minutes by tapping the tube with your finger.
  • Antibody Dilutions: While default ratios are listed (1:99 for primary, 1:50 for secondary), these depend heavily on your specific cell line and antibody; always titer your antibodies if necessary.

Instrument Operation

  • Calibration Control: Always prepare and include a separate FACS tube containing 350 µL of untreated cells in PBS to calibrate the instrument.
  • Filter Selection: Correctly designate your fluorescent dye within the software so the instrument applies the appropriate optical filter.
  • Run Order: Begin your sample run with the non-treated cells first, followed by the positive cells to clearly distinguish the experimental difference.
  • Flow Speed: Operating the instrument at "low" speed is best, though "medium" and "high" speeds are acceptable depending on your sample size.
  • Post-Run Maintenance: Thoroughly clean the instrument immediately after finishing your run by following the specific instruction paper pinned next to the machine.


Materials
Cell Source: Cultured cells in 10 cm Petri dishes.
Buffers & Dissociation Solutions: Phosphate-Buffered Saline (PBS) – prepare both room temperature and ice-cold stocks. Versene or Trypsin.
Antibodies: Primary antibody specific to your target surface receptor. Fluorescently-conjugated secondary antibody.
Pipetting: 5 mL and 10 mL glass pipettes paired with a pipette boy.
Tubes: Standard Falcon tubes and specialized glass FACS tubes.
Quantification: Cell counting chamber slide and a laboratory microscope.
Instruments: Vortex mixer, centrifuge, and a flow cytometer (such as the BD FACSymphony A1).
Temperature Control: Ice bucket. Critical Preliminaries
Safety warnings
Critical: Temperature Control
You must use ice-cold PBS for the 8 mL resuspension step and for all subsequent washes from step 7 onward. Maintaining an ice-cold temperature is absolutely critical to halt endocytosis. If the buffer warms up, surface receptors can internalize the antibodies, compromising your cell surface tagging.

Safety: Vortexing Check
Before vortexing your resuspended cell sediment in the Falcon tube, inspect the plastic closely to ensure the tube is not brittle, avoiding tube failure and sample loss.

Handling: Avoid Vortexing During Washes
When washing out the primary and secondary antibodies, resuspend the cell pellets well but do not vortex.

Instrument Safety: Cap Removal
Always remove the tube cap before placing your specialized glass FACS tube onto the cytometer instrument loader.
Before start
Prepare Ice: Fill an ice bucket before beginning. From step 7 onward, the use of ice-cold PBS and keeping samples on ice is mandatory. This temperature control halts endocytosis, preventing the internalization of antibodies when targeting surface receptors. Antibody Titration: While this protocol notes a default baseline dilution of 1:99 for the primary antibody and 1:50 for the secondary antibody, these ratios depend heavily on your specific cell line and antibody lot; independent titration may be necessary before execution. Experimental Controls: Remember to set aside a dedicated aliquot of untreated cells (350 µL in PBS) to serve as a negative control for instrument calibration and gating. Safety Check: Inspect your Falcon tubes before vortexing to ensure they are not brittle, minimizing the risk of tube breakage and sample loss.
Prepare the cells from 10 cm Petri dishes
30m
Wash the cells with 5 mL of PBS two times (2x)

Note:
Room tempearture
Mix gently
Use the 5 mL glass pipettes with the pipette boy
2m
Wash the cells with 2 mL of versene or trypsin or PBS two times (2x)

Note:
Room tempearture
Mix gently
Use the 10 mL glass pipettes with the pipette boy
5m
Add 8 mL of ice-cold PBS* and resuspend

Note:
Use 10 mL glass pipettes with the pipette boy
*It needs to be ice-cold PBS to stop endocytosis. This is important when tagging surface receptors so that the antibodies don't internalise into the cell
1m
Transfer to a falcon tube and centrifuge @1000 RPM for 6 minutes

Note:
Room temperature
6m
Remove supernatant and resuspend the sedimented cells in 10 mL of ice-cold PBS

Mix very well so the cells dilute. Can use vortex. Before vortex, check that the Falcon tube is not brittle
1m
Vortex well and add 10 μL of the cell suspension into one cell counting chamber (Figure 1).

Figure 1. Cell counter chambers

Count the cells on a plate counter slide under a microscope. Count in five large squares in "zig-zag" pattern. Start from any top square (Figure 2)

Figure 2. Cell counting in chambers
After use, note that the chamber was used by writing an "x" on it

Calculate the cell concentration with a formula:

c(cells/mL): cell concentration
Ncells: Total counted number of cells in all four squares
10m
Dilute in PBS 5 x 105 cells/50 μL (=107 cells/mL)

Calculate the amount that is needed for the experiment, centrifuge @1200 RPM, 5 min and resuspend in the right amount of ice cold PBS
From now on use ice cold PBS for all washes. Cold PBS prevents endocytosis and therfore internalization of the antiboties inside the cells. This is very important when targeting surface receptors.
5m
Binding of the primary antibody
1h
Add primary antibody in dilution 1:99* (1 volume antibody to 99 volumes cell suspension). It can be added 1 μL of primary antibody to 99 μL of cells

Leave it 1 hour on ice. Every 15 minutes gently mix the solution by tapping it with finger

*The ratio will depend on the antibody and the cell line used. Some antibodies need to be tittered
1h
Binding of the secondary antibody and prep for FACS
40m
Wash out the primary antibody with 450 μL of ice-cold PBS:

1. Add 450 μL of ice-cold PBS and resuspend the cells well - do not vortex
2. Centrifuge down the cells @1200 RPM, 5 min
3. Remove supernatant and add 450 μL of ice-cold PBS
4. Centrifuge down the cells @1200 RPM, 5 min
5. Remove supernatant and add 98 μL of ice-cold PBS
15m
Add 2 μL of secondary antibody (dilution 1:50*) to the cell suspension in ice-cold pBS

Leave it 1 hour on ice. Every 15 minutes gently mix the solution by tapping it with finger

*The ratio will depend on the antibody and the cell line used. Some antibodies need to be tittered
Wash out the secondary antibody with 450 μL of ice-cold PBS:

1. Add 450 μL of ice-cold PBS and resuspend the cells well - do not vortex
2. Centrifuge down the cells @1200 RPM, 5 min
3. Remove supernatant and add 450 μL of ice-cold PBS
4. Centrifuge down the cells @1200 RPM, 5 min
5. Remove supernatant and add 350 μL of ice-cold PBS
15m
Resuspend the cells well and transfer 350 μL into the special glass tubes (Facs tubes, Figure 3)

Figure 3. A representation of a Facs tube used for flow cytometry

Remember to also include 350 μL of untreated cells in PBS for calibration of the device in one of the Facs tubes
10m
FACS
1h 25m
Follow the instrument instructions on the instrument (Figure 4)


Figure 4. The Flow cytometer used in the lab

Choose your fluorescent dye so the instrument knows which filter to put
5m
Start the run with the non-treated cells, followed by positive cells to clearly see the difference

The Facs tube is put onto the instrument after removing the tube cap

The instrument has low, medium and high speed settings, best is going with low, but depending on the sample sizes, it is acceptable medium and high speeds
1h
After finishing the run, clean well the instrument. The instructions are clearly noted on a paper pinned next to the machine.
20m
Data analysis
Analyse the fluorescence and cell data on the computer
Acknowledgements
Prepared by Dr. sc. Bruno Pavletić, Dr. sc. Mario Stojanović, M.sc. Ana Runtić, M.sc. Roberto Galov & Dr. sc. Dragomira Majhen*

*Senior researcher and corresponding author