Standard Flow Cytometry Staining (Surface, Intracellular, Intranuclear)

Dillon Corvino

Standard workflow for surface, intracellular, and intranuclear staining of cells for flow cytometry, including optional tetramer staining performed together with surface antibodies.
Published

December 30, 2025

Protocol ID: GEN-FLOW-STD
Version: v1.4
Author: Dillon Corvino

Purpose

This protocol describes a standard flow cytometry staining workflow for suspension cells, including:

  • Surface staining with Fc block and live/dead dye
  • Optional tetramer staining performed together with surface staining
  • Optional intracellular staining (e.g. BD Cytofix/Cytoperm-based)
  • Optional intranuclear staining (e.g. eBioscience FOXP3 fix/perm-based)

It is intended as a general framework for murine or human samples and can be adapted to specific panels and antibody combinations.

Buffer nomenclature (important)

  • FACS buffer = PBS + FCS
  • MACS buffer = PBS + FCS + EDTA

Unless otherwise specified, MACS buffer is used throughout this protocol to minimise cell clumping during staining and washing.

When tetramers are included, all surface staining (including tetramers and live/dead dye) is performed in FACS buffer (no EDTA).

Critical notes (read before starting)

  • Keep cells and antibody mixes cold (on ice or at 4 °C) and protected from light whenever possible.
  • Default staining range: 2 × 10⁵ – 1 × 10⁶ cells per well (96-well format).
  • Always include appropriate controls (unstained, live/dead only, FMO, single-colour controls).
  • Fixation and permeabilization conditions must be compatible with antibodies and fluorochromes used.
  • Commercial fixation/permeabilization kits should be used according to manufacturer instructions.
Warning

Improper fixation/permeabilization or incompatible buffers can destroy epitopes and fluorochromes, leading to misleading intracellular or intranuclear staining.

Warning

Antibodies and tetramers are protein reagents and mechanically sensitive.
Do not vortex, as this can denature proteins and promote aggregation.
Instead, mix gently by pipetting or inversion. If needed, briefly spin reagents in a mini “pulse” centrifuge (quick spin) to collect liquid and pellet aggregates before use.

Tip

Surface markers (including live/dead dyes and tetramers) should generally be stained before fixation/permeabilization for best performance.

Tip

Surface staining master mixes can be prepared in advance (e.g. the day before) to save time; however, it is recommended to add the live/dead dye immediately before use.

Approximate timing

  • Surface ± tetramer staining: 30 min
  • Plate setup and Fc block: 10–15 min
  • Fixation only (no intracellular): 15–20 min
  • Fix + permeabilization + intracellular/intranuclear staining: 1.5–2 h
  • Optional overnight intranuclear stain: +1 day

Total (surface only): ~1–1.5 h
Total (full workflow): ~3–4 h (or overnight)

Procedure

Step 1 – Plate setup and buffers

  1. Prepare buffers:
    • FACS buffer (PBS + 2% FCS) — use only if tetramers are included
    • MACS buffer (PBS + 2% FCS + 2 mM EDTA) — default buffer
  2. Prepare single-cell suspensions.
  3. Count cells and adjust concentration to plate 2 × 10⁵ – 1 × 10⁶ cells per well.
  4. Aliquot cells into a 96-well U- or V-bottom plate.
  5. Keep plates on ice or at 4 °C.

Step 2 – Surface staining (with live/dead ± tetramers)

Surface staining is performed in a single master mix.

  • If tetramers are included → use FACS buffer (no EDTA)
  • If no tetramers are included → use MACS buffer

Antibody titration guidance (applies to all surface staining): - Surface antibodies: default 1:200 if titration data are unavailable. - Transcription factors or weakly expressed markers: default 1:50.

2.1 Prepare surface staining master mix

Prepare a 1× surface staining master mix in the appropriate buffer containing:

  • Fc block: 1:100
  • Live/dead dye (1× final concentration)
  • Surface antibodies (1× final concentration)
  • Optional: Tetramer(s), per manufacturer recommendation

Final volume: 50 µL per well

Note

When tetramers are included, staining is performed in FACS buffer without EDTA to avoid potential interference with multimer–TCR interactions.
Whether EDTA exclusion is strictly necessary remains under investigation and will be empirically evaluated within the lab.

Tip

After flicking off the supernatant, residual liquid in the dead volume can be used to resuspend cell pellets by briefly vortexing the plate at low speed to agitate the wells. This helps achieve uniform resuspension before adding the next reagent.

2.2 Staining

  1. Resuspend each cell pellet in 50 µL surface staining master mix.
  2. Incubate for 30 min on ice, protected from light.

2.3 Wash steps

  1. Top up each well to 200 µL with MACS buffer.
  2. Centrifuge at 1000 g for 2 min at 4 °C.
  3. Flick off the supernatant.
  4. Wash once more with 200 µL MACS buffer, centrifuge at 1000 g for 2 min at 4 °C, and flick.
Note

All antibodies and tetramers should be titrated for optimal performance. Default dilutions are provided as a starting point only.

2.4 Surface staining only

  1. Resuspend pellets in 100–200 µL 4% PFA in PBS.
  2. Wrap the plate in aluminium foil to protect from light and clearly label with name and date.
  3. Store at 4 °C, protected from light, until acquisition.

Step 3 – Fixation and permeabilization

This step assumes use of: - BD Cytofix/Cytoperm (cytoplasmic targets), or
- eBioscience FOXP3 fix/perm (intranuclear targets).

3.1 Initial fixation/permeabilization

  1. Add 100 µL fix/perm solution per well.
  2. Incubate 20 min on ice, protected from light.
  3. Top up to 200 µL with 1× PermWash.
  4. Centrifuge at 1000 g for 2 min at 4 °C and flick.
  5. Resuspend in 200 µL PermWash.
  6. Centrifuge at 1000 g for 2 min at 4 °C and flick.

3.2 Optional pause point

  1. Resuspend pellets in 200 µL MACS buffer.
  2. Wrap the plate in aluminium foil to protect from light and clearly label with name and date.
  3. Store at 4 °C overnight, protected from light.
Note

If the protocol is paused at this step, the recovery steps below must be performed before proceeding to intracellular or intranuclear staining.

3.3 Restarting the protocol after pause point

  1. Centrifuge the plate at 1000 g for 2 min at 4 °C.
  2. Flick off the supernatant.
  3. Resuspend pellets in 200 µL PermWash.
  4. Incubate for 30 min on ice, protected from light.
  5. Centrifuge at 1000 g for 2 min at 4 °C and flick.
  6. Proceed directly to Step 4 – Intracellular or intranuclear staining.

Step 4 – Intracellular or intranuclear staining

  1. Prepare an intracellular or intranuclear antibody master mix in the appropriate PermWash buffer (kit-specific):
    • Final volume: 50 µL per well
  2. Resuspend each cell pellet in 50 µL of the antibody master mix.
  3. Incubate 30 min on ice, protected from light, or incubate overnight at 4 °C.
Tip

For intranuclear targets (e.g. FOXP3, Helios, T-bet, Eomes), overnight antibody incubation at 4 °C can improve signal intensity, particularly for low-abundance or epitope-masked transcription factors.
Ensure plates are well sealed, clearly labelled, and protected from light during overnight incubation.

Warning

Overnight antibody incubation is not recommended for cytoplasmic intracellular targets (e.g. cytokines, granzymes, perforin). Prolonged incubation can increase background staining and does not typically improve signal for these antigens. Use 30–60 min incubations for intracellular (non-nuclear) targets.

  1. Top up each well to 200 µL with PermWash.
  2. Centrifuge at 1000 g for 2 min at 4 °C and flick off the supernatant.
  3. Wash once more with 200 µL PermWash, centrifuge at 1000 g for 2 min at 4 °C, and flick.
  4. Resuspend each pellet in 100–200 µL 4% PFA in PBS.
  5. Wrap the plate in aluminium foil to protect from light and clearly label with name and date.
  6. Store at 4 °C, protected from light, until acquisition.

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

None

Materials

Reagents

Reagent Notes
PBS 1× Ca²⁺/Mg²⁺-free
FCS (heat-inactivated) 2% v/v
EDTA 2 mM (MACS buffer only)
Fc block Mouse: anti-CD16/32
Live/dead dye As per manufacturer
Tetramers (e.g. MR1–5-OP-RU, CD1d) Optional
Surface antibodies Titrate individually
Intracellular/intranuclear antibodies Kit-compatible
Fix/perm kits BD or eBioscience
4% PFA Fixation

Version history

Version Date Author Change summary
v1.0 2025-11-21 Dillon Corvino Initial version
v1.1 2025-12-29 Dillon Corvino Added tetramer workflow, clarified buffers, titration defaults, overnight intranuclear staining
v1.2 2025-12-30 Dillon Corvino Unified tetramer and surface staining into single master mix; buffer choice conditional on tetramer inclusion
v1.3 2025-12-30 Dillon Corvino Added conditional restart steps after fix/perm pause point and explicit plate centrifugation conditions
v1.4 2026-09-18 Dillon Corvino Replaced # Buffers used (bare library(tidyverse), leaked R package-startup warnings) with an auto-generated # Buffers Required section; moved it from after Materials to before Materials