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Method Article

Flow Cytometric Detection of Intracellular Proteins in K562 Human Erythroleukemia Cells Using Unconjugated Primary Antibodies

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DOI:

10.3791/72500

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September 8th, 2026

 ,  , 
1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona

In This Article

Summary

This protocol describes flow cytometric detection of intracellular proteins in K562 human erythroleukemia cells using unconjugated primary polyclonal antibodies. Antibody specificity is demonstrated by dose-dependent inhibition of staining through peptide competition.

Abstract

Flow cytometry is a powerful technique for quantitative analysis of protein expression at single-cell resolution. This protocol describes a workflow for detecting intracellular proteins in K562 human erythroleukemia cells using unconjugated primary polyclonal antibodies and fluorophore-conjugated secondary antibodies. Cells are sequentially fixed, permeabilized, and blocked before antibody staining. By varying the order of antibody incubation relative to fixation and permeabilization, the protocol enables assessment of proteins with surface or intracellular localization using separate staining workflows. Nuclear staining of splicing factor 3a subunit 1 (SF3A1) is demonstrated using an affinity-purified rabbit anti-SF3A1 polyclonal antibody, and antibody specificity is evaluated by dose-dependent inhibition of staining with the corresponding antigenic peptide. In addition, staining conditions are used to assess reactivity of an antibody targeting the intracellular domain of erythropoietin receptor. This standardized workflow expands the use of unconjugated primary antibodies in flow cytometry and provides a practical approach for evaluating protein expression and localization, as well as antibody specificity in K562 human erythroleukemia cells.

Introduction

Flow cytometry is a powerful and widely used analytical tool that allows quantitative assessment of protein expression at single-cell resolution, thereby facilitating examination of heterogeneous and rare cell populations1,2,3. While conventional flow cytometry approaches primarily employ antibodies targeting cell surface antigens, many functionally important proteins, such as transcription factors, RNA-processing regulators, and receptors that undergo ligand-induced internalization, are localized within intracellular compartments. Consequently, these cytoplasmic and nuclear proteins are inaccessible using standard surface staining protocols. The incorporation of intracellular staining methods, which involve fixation and permeabilization of cellular membranes, has substantially expanded the utility of flow cytometry by allowing quantitative assessment of intracellular proteins, cytokines, and signaling intermediates while preserving the high-throughput and multiparametric capabilities of conventional flow cytometry analysis4,5,6,7.

A key limitation in detecting both surface-associated and intracellular proteins by flow cytometry is the availability of suitable antibodies and, in many cases, incomplete knowledge of epitope or immunogen sequences8,9. Since immunophenotyping strategies are largely based on cell surface markers, most commercially available antibodies validated for flow cytometry are monoclonal, recognize single extracellular epitopes, and are preconjugated to fluorophores. In contrast, polyclonal antibodies are capable of recognizing multiple epitopes within a target protein and are commonly used for western blotting, immunohistochemistry, or immunofluorescence. However, these antibodies are typically unconjugated and therefore not directly compatible with standard flow cytometry workflows. The use of unconjugated primary antibodies in combination with fluorophore-conjugated secondary antibodies provides a flexible alternative that expands the range of detectable targets and may enable signal amplification, which can be advantageous for low-abundance proteins.

To address these limitations, we optimized a protocol for staining proteins in K562 human erythroleukemia cells10. Intracellular staining requires fixation to preserve cellular structure and state, as well as permeabilization to allow antibodies to access intracellular compartments. In this protocol, cells are fixed with paraformaldehyde (PFA), a commonly used crosslinking fixative that maintains cellular morphology and may increase cell membrane permeability under specific conditions11,12,13,14. Permeabilization with ethanol (EtOH) allows antibody entry into intracellular compartments, including access to nuclear targets5,13,15. Permeabilization is followed by staining with unconjugated primary polyclonal antibodies and fluorophore-conjugated secondary antibodies. Using this workflow, PFA treatment alone is used for detection of non-surface immunoreactivity, whereas an additional EtOH permeabilization step is used for nuclear target detection. Assessment of nuclear staining specificity by peptide competition showed dose-dependent inhibition of antibody binding by the cognate peptide and not by a nonspecific control. Overall, the use of unconjugated primary polyclonal antibodies extends the applicability of flow cytometry beyond conventional surface marker analysis with fluorophore-conjugated antibodies and provides a flexible approach for assessing intracellular protein expression at single-cell resolution.

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Protocol

K562 cells were obtained commercially and were periodically authenticated and tested for mycoplasma by the University of Arizona Genetics Core. No human participants or vertebrate animals were involved in this study.

1. Prepare reagents

  1. Culture cells under sterile conditions. Use the K562 human myelogenous leukemia cell line for this protocol.
    1. Thaw and maintain K562 cells in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO₂16. For propagation, seed cells at a density of approximately 1 × 105 cells/mL, harvest when cultures reach 75%–95% of the recommended maximum cell density, and do not culture beyond 20 passages. For staining, harvest cells at 50%–75% of the recommended maximum cell density.
  2. Prepare sterile 10× phosphate-buffered saline (PBS) inside a class II biosafety cabinet.
    1. Add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 2.4 g potassium phosphate monobasic (KH₂PO₄), and 14.4 g sodium phosphate dibasic (Na₂HPO₄) to 800 mL autoclaved water in a 1 L graduated cylinder.
    2. Add a stir bar and stir the solution on a magnetic stir plate until all powder has dissolved.
    3. Use a pH probe to adjust the pH to 7.2–7.4. Bring the final volume to 1 L with autoclaved water. Filter-sterilize the solution using a bottle-top filter fitted with a 0.2 µm pore-size nylon membrane.
  3. Dilute 10× PBS to 1× PBS by adding 100 mL sterile 10× PBS to 900 mL autoclaved water inside a class II biosafety cabinet.
  4. Prepare the fixable viability dye according to the manufacturer's instructions. For this protocol, add 50 µL dimethyl sulfoxide (DMSO) to one tube of lyophilized dye and vortex until the dye is completely dissolved.
    1. Store the reconstituted dye at −20°C, protected from light, for up to 1 year. Store the dye in small aliquots to avoid repeated freeze-thaw cycles.
  5. Prepare fluorescence-activated cell sorting (FACS) buffer by combining 10% 10× PBS, 86% water, and 4% fetal bovine serum (FBS). For example, combine 10 mL 10× PBS, 86 mL water, and 4 mL FBS to prepare 100 mL FACS buffer.
    1. Prepare only the volume of FACS buffer needed for approximately 2 weeks.
    2. Store FACS buffer at 4°C.
  6. Prepare 1% paraformaldehyde (PFA).
    CAUTION: Handle PFA as a flammable, reactive carcinogen. Wear appropriate personal protective equipment and prepare PFA inside a certified chemical fume hood.
    1. Mix 350 mL water and 50 mL 10× PBS, place the solution on a heated stir plate, and heat it to 65°C. Gradually add 20 g PFA powder to the heated solution to prepare 4% PFA (weight/volume).
    2. Add 1–3 drops of 5 M sodium hydroxide (NaOH) to facilitate dissolution of the PFA. Stir the solution on a heated stir plate for approximately 20 min. Use a pH meter to adjust the pH to 7.4.
    3. Bring the final volume to 500 mL with water.
    4. Aliquot the 4% PFA into 50 mL volumes and store at −20°C for up to 1 year. After thawing, store the 4% PFA at 4°C for up to 2 months. Discard any remaining thawed 4% PFA after 2 months.
    5. Dilute the 4% PFA to 1% PFA using 1× PBS and store the diluted solution at 4°C for up to 4 weeks17.
  7. Prepare cold 70% ethanol (EtOH) by combining 70 mL of 200-proof ethanol with 30 mL of Type I ultrapure water.
    1. Store the 70% EtOH at −20°C for up to 1 year. Keep the solution ice cold immediately before addition to cells for permeabilization.

2. Collect and count cells

  1. Transfer cells from a 10 cm tissue culture plate into a 15 mL centrifuge tube.
  2. Centrifuge the cells at 600 × g for 5 min at 4°C with the brake on to pellet the cells.
  3. Remove the culture medium and wash the cells by resuspending them in 5 mL of 1× PBS. Centrifuge again at 600 × g for 5 min at 4°C with the brake on, then aspirate the supernatant, leaving the cell pellet.
  4. Resuspend the cell pellet in 1× PBS. Count the cells using a hemocytometer or an automated cell counter by mixing 10 µL of cell suspension with 10 µL trypan blue, loading the sample onto a cell counting slide, and determining the cell concentration.
    NOTE: Proceed only if cell viability is >90% to ensure reliable results.
  5. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with brake on and aspirate the supernatant, leaving the cell pellet. Resuspend the cells to a density of 2 × 105 viable cells/mL (minimum 0.5 × 105 viable cells/mL).
  6. Dispense 1 mL of cell suspension into the required number of sample and control flow tubes.
    1. Prepare separate sample tubes for each target protein and staining workflow. Surface (Pre-PFA), Post-PFA, Pre-EtOH, and Post-EtOH staining conditions are performed using independent sample tubes rather than sequential staining of the same cells. For example, when assessing two target proteins, prepare eight tubes: Tube 1 (primary antibody A, Pre-PFA), Tube 2 (primary antibody B, Pre-PFA), Tube 3 (primary antibody A, Post-PFA), Tube 4 (primary antibody B, Post-PFA), Tube 5 (primary antibody A, Pre-EtOH), Tube 6 (primary antibody B, Pre-EtOH), Tube 7 (primary antibody A, Post-EtOH), and Tube 8 (primary antibody B, Post-EtOH).
      NOTE: As shown in the staining workflow (Figure 1), perform primary and secondary antibody staining for the Pre-PFA and Pre-EtOH samples on Day 1. Perform primary and secondary antibody staining for the Post-PFA and Post-EtOH samples on Day 2.
    2. Prepare unstained and single-stained control tubes for each treatment condition. If assessing viability and two target proteins, prepare one unstained control, one viability dye-only control, and one secondary antibody-only control for each treatment condition.
      NOTE: Although the primary antibody is unconjugated, a primary antibody-only control may be included during assay validation or when evaluating nonspecific primary antibody binding.
    3. Subject all unstained and single-stained control tubes to the same treatments as the sample tubes. If samples are fixed with 1% PFA and permeabilized with 70% EtOH, process the control tubes identically.
  7. Prepare single-stained compensation controls using compensation beads.
    NOTE: Beads may be used instead of cells to prepare compensation controls.
    1. Label one compensation control tube for each fluorophore and one unstained control tube. If using a viability dye and two secondary antibodies labeled with different fluorophores, prepare four tubes: one unstained control, one viability dye control (fluorophore 1), one secondary antibody A control (fluorophore 2), and one secondary antibody B control (fluorophore 3).
      NOTE: If using an amine-reactive viability dye, use amine-reactive compensation beads for the viability dye control.
    2. Add only one dye or antibody to each compensation control tube so that each control is positive for a single fluorophore during flow cytometric compensation.

Antibody staining protocol chart with pre/post-fixation steps, outlining sequential processes.
Figure 1. Optimized staining workflow for surface-associated and intracellular target detection. The workflow illustrates the sequence and timing of fixation, permeabilization, blocking, primary antibody staining, and secondary antibody staining following fixable viability dye staining. Separate workflows are shown for the Pre-PFA, Post-PFA, Pre-EtOH, and Post-EtOH conditions. Primary and secondary antibody staining are performed on Day 1 for the Pre-PFA and Pre-EtOH workflows and on Day 2 for the Post-PFA and Post-EtOH workflows. PFA, paraformaldehyde; EtOH, ethanol. Please click here to view a larger version of this figure.

3. Stain cells with a fixable viability dye

  1. Stain the cells with a fixable viability dye according to the manufacturer’s instructions. Add 1 µL of viability dye stock solution (prepared in Step 1.4) directly to each sample tube and the corresponding viability dye single-stained control tube containing 1 × 106 cells in 1 mL of 1× PBS.
  2. Vortex gently for 3 s and incubate the samples at room temperature for 30 min, protected from light.
    NOTE: Protect the cells from light during this and all subsequent steps.
  3. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on, then aspirate the supernatant, leaving the cell pellet.
  4. Wash the cells twice with 1 mL of 1× PBS and resuspend the final cell pellet in 100 µL of 1× PBS.
    NOTE: If staining cell surface targets with fluorophore-conjugated primary antibodies, perform surface staining at this stage according to the manufacturer's instructions before proceeding to fixation.

4. Treat cells with 1% PFA

NOTE: For Pre-PFA samples (see Step 2.6.1 and Figure 1), perform Steps 6, 7, and 8 before proceeding with this section.

  1. To the cells resuspended in 100 µL of 1× PBS, add 1 mL of 1% PFA to each sample tube, including all single-stained control tubes. Vortex gently for 3 s and incubate the samples at room temperature for 15–20 min, protected from light.
  2. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on. Aspirate and discard the supernatant, leaving the cell pellet.
  3. Wash the cell pellet twice by resuspending it in 1 mL of FACS buffer, followed by centrifugation at 600 × g for 5 min at 4°C with the brake on.
  4. Resuspend the final cell pellet in 100 µL of FACS buffer.
    ​NOTE: Fixed cells (stained or unstained) may be stored in 1 mL of FACS buffer at 4°C for 12–48 h. To resume the protocol, centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on, aspirate the supernatant, and resuspend the cell pellet in 100 µL of FACS buffer.

5. Permeabilize cells with EtOH

NOTE: For Pre-EtOH samples (see Step 2.6.1 and Figure 1), perform Steps 6, 7, and 8 before proceeding with this section.

  1. Gently add 1 mL of ice-cold 70% EtOH to the inside wall of each sample tube containing 100 µL of cell suspension, then immediately vortex at approximately 50% power for 5 s. Repeat this procedure for all remaining sample tubes and single-stained control tubes.
  2. Incubate the tubes at −20°C for at least 2 h, protected from light.
    ​NOTE: Cells may be stored in 1 mL of 70% EtOH at −20°C for 12–48 h before continuing the protocol.
  3. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on, then aspirate the supernatant, leaving the cell pellet.
  4. Wash the cells three times with 1 mL of FACS buffer, centrifuging at 600 × g for 5 min at 4°C with the brake on between washes.
  5. Resuspend the final cell pellet in 100 µL of FACS buffer.

6. Block cells

  1. Add 820 µL of FACS buffer and 80 µL of normal goat serum (NGS) to each tube to prepare a final blocking solution containing 92% FACS buffer and 8% NGS. Vortex gently for 3 s and incubate the samples at room temperature for 30 min, stationary and protected from light.
  2. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on, then aspirate the supernatant, leaving the cell pellet.
  3. Wash the cells twice by resuspending the pellet in 1 mL of FACS buffer followed by centrifugation as described in Step 6.2.
  4. Resuspend the final cell pellet in 100 µL of FACS buffer.

7. Stain cells with an unconjugated primary antibody

  1. Add 0.5 µL of the unconjugated primary antibody to 100 µL of cell suspension (1:200 dilution). Incubate the samples at room temperature for 1 h, protected from light.
    NOTE: The 1:200 dilution is a recommended starting concentration and may require optimization for other antibodies.
    1. If performing peptide competition, add the peptide immediately before adding the primary antibody. In this protocol, peptides were titrated at peptide-to-antibody molar ratios ranging from 50 to 0.625, calculated relative to the antibody concentration (4.3 nM) in the reaction mixture. Optimization may be required for individual peptide–antibody pairs.
  2. Add 1 mL of FACS buffer and centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on. Aspirate the supernatant, leaving the cell pellet.
  3. Wash the cells twice more with 1 mL of FACS buffer, centrifuging as described in Step 7.2.
  4. Resuspend the final cell pellet in 100 µL of FACS buffer.

8. Stain cells with a fluorophore-conjugated secondary antibody

  1. Add a 1:200 dilution of the fluorophore-conjugated secondary antibody (0.5 µL antibody in 100 µL of cell suspension) to each sample tube and the corresponding control tube.
    NOTE: Goat anti-rabbit DyLight 488 secondary antibody was used in this protocol.
  2. Incubate the samples at room temperature for 1 h, protected from light.
  3. Add 1 mL of FACS buffer. Centrifuge the cell suspension at 600 × g for 5 min at 4°C with the brake on, then aspirate the supernatant, leaving the cell pellet.
  4. Wash the cells twice with 1 mL of FACS buffer by repeating the centrifugation described in Step 8.3.
  5. Resuspend the cells in 300–500 µL of FACS buffer.
    NOTE: Cells may be stored in FACS buffer at 4°C, protected from light, for 1–3 days before flow cytometric analysis.

9. Analyze samples by flow cytometry

  1. Perform fluorescence compensation using the appropriate single-stained controls. Acquire the samples on a flow cytometer and collect at least 5,000–10,000 live single-cell events for analysis. For this protocol, FACSDiva software was used for data acquisition.
  2. Analyze the data by first excluding debris using forward scatter area (FSC-A) versus side scatter area (SSC-A), followed by exclusion of doublets using FSC-height (FSC-H) versus FSC-width (FSC-W) and SSC-height (SSC-H) versus SSC-width (SSC-W). Exclude dead cells using the viability dye channel.
  3. Analyze the percentage of the remaining live single cells that are positive or negative for the selected target proteins (see Representative Results). Establish positive gates using the unstained and secondary antibody-only controls, and apply identical gating criteria to all comparable samples within the same acquisition batch.

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Results

The protocol described here enables intracellular staining of protein targets for which commercial antibodies validated for flow cytometry are unavailable. As a representative example, staining of splicing factor 3a subunit 1 (SF3A1), a ubiquitously expressed splicing protein that localizes to the nucleus, was evaluated using a rabbit polyclonal antibody raised against a peptide corresponding to amino acids 18–37 of SF3A1 (SF3A118–37; EPKQPTEEEASSKEDSAPSK)18,

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Discussion

The results presented here highlight several critical parameters for the detection of proteins by intracellular flow cytometry using unconjugated primary antibodies. An important consideration is the permeabilization strategy, which should be selected according to the intracellular localization of the target protein. In the workflow described here, PFA fixation followed by EtOH permeabilization enabled detection of the nuclear protein SF3A1, whereas EpoR immunoreactivity was detected using both the Post-PFA and Post-EtOH...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge funding support to SS from the National Institute of General Medical Sciences (R01GM127464) and the National Cancer Institute (P30CA023074), both of the National Institutes of Health, the Valley Research Partnership Program (VRP P1-4009 and VRP77), and the Arizona Biomedical Research Center (ABRC: RFGA2022-010-30). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors also acknowledge the support of Dr. Mrinalini Kala, Director of the Flow Cytometry Core Facility, College of Medicine–Phoenix, University of Arizona.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10× Phosphate-buffered saline (PBS)In-houseN/AAlternative: Commercially available PBS
15 mL centrifuge tubesVWR89039-664Alternative: Any sterile centrifuge tube of appropriate size
5 mL round-bottom polystyrene tubesCorning352052Alternative: Any flow cytometry tube compatible with the available cytometer
Anti-rabbit DyLight 488 secondary antibodyThermo Fisher ScientificPI35553Alternative: Secondary antibody compatible with the primary antibody host species
Automated cell counterThermo Fisher ScientificAMQAF2000Alternative to hemocytometer
ArC Amine Reactive Compensation Bead KitThermo Fisher ScientificA10628Alternative: Cells; if using cells for single-stain controls, process all control tubes identically to sample tubes
Bottle-top filterThermo Fisher Scientific595-4520Nylon membrane; 0.2 µm
Cell counting slidesThermo Fisher ScientificC10228Required only when using an automated cell counter
Dimethyl sulfoxide (DMSO)Supplied with viability dye kitN/AUsed to reconstitute the lyophilized viability dye
Ethanol, 200 proofThermo Fisher ScientificBP28184Molecular biology grade; used to prepare ice-cold 70% ethanol
Erythropoietin Receptor Primary Polyclonal AntibodyBiossBS-1424R
Fetal bovine serum (FBS)Omega ScientificFB-12Alternative: Any FBS validated for the target cell line
Flow cytometerBD BiosciencesBD Canto II Alternative: Any suitable flow cytometer
HemocytometerHausser ScientificHS3200Alternative: Automated cell counter
Heated magnetic stir plateThermo Fisher ScientificSP88857100Used for preparation of PBS and paraformaldehyde
K562 human myelogenous leukemia cellsATCCCCL-243Can purchase from any other authenticated source
Live-or-Dye fixable viability dye (405/545)Biotium32009Alternative: Any compatible fixable viability dye. Non-fixable dyes (e.g., 7-AAD) are not suitable
Normal goat serumThermo Fisher Scientific16210064Used for blocking
Paraformaldehyde powderSigma-AldrichP6148Alternative: Commercially available paraformaldehyde solution
pH meter/probeAccumetAB15Used to adjust buffer pH
Potassium chloride (KCl)Thermo Fisher ScientificBP366-1
Potassium phosphate monobasic (KH2PO4)Thermo Fisher ScientificBP362-1
SF3A1  Primary Polyclonal AntibodyIn-houseN/A
Sodium chloride (NaCl)Thermo Fisher ScientificBP358-1
Sodium hydroxide solution (5 M)MilliporeSigmaS8263Alternative: Prepare a 5 M solution from sodium hydroxide pellets
Sodium phosphate dibasic (Na2HPO4)Thermo Fisher ScientificBP332-1
Stir barVWR74950-286Used with the magnetic stir plate
Trypan Blue, 0.4%Thermo Fisher Scientific15250061Used for cell viability counting
UltraComp eBeads Compensation BeadsThermo Fisher Scientific01-2222-42Alternative: Cells; if using cells for single-stain controls, process all control tubes identically to sample tubes

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