May 11, 2026
Flow Cytometry Protocol for Human and Mouse CTLA-4 (CD152) Detection: Surface and Intracellular Staining
- abinScience cheng1
- 1abinScience, AtaGenix Laboratories, Wuhan, China
External link: https://www.abinscience.com
Protocol Citation: abinScience cheng 2026. Flow Cytometry Protocol for Human and Mouse CTLA-4 (CD152) Detection: Surface and Intracellular Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dwr3g1y/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: May 09, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316667
Keywords: flow cytometry protocol for human, flow cytometry, flow cytometry protocol, intracellular staining procedure, cell surface, cd152
Disclaimer
For research use only. Not for diagnostic or therapeutic purposes.
Abstract
This protocol describes surface and intracellular staining procedures for detecting CTLA-4 (CD152) expression on human and mouse T cells by flow cytometry. Unlike most surface markers, CTLA-4 is predominantly stored in intracellular vesicles and only transiently expressed on the cell surface upon T cell activation. Therefore, this protocol covers both surface staining for activated T cells and intracellular staining for total CTLA-4 detection, particularly in regulatory T cells (Tregs). Optimized for use with abinScience research-grade anti-CTLA-4 monoclonal antibodies in multiple conjugated formats (FITC, PE, APC, PerCP).
Cell Preparation and T Cell Activation
Cell Preparation and T Cell Activation
For surface CTLA-4 detection, T cells MUST be activated prior to staining, as resting T cells express negligible surface CTLA-4.
1. Isolate PBMCs from whole blood by density gradient centrifugation (Ficoll-Paque, 400 × g, 30 min).
2. For activated T cells: stimulate PBMCs with anti-CD3/CD28 beads or plate-bound anti-CD3 (1 μg/mL) + soluble anti-CD28 (2 μg/mL) for 48–72 hours at 37°C, 5% CO₂.
3. For Treg analysis (intracellular CTLA-4 only): activation is not required — freshly isolated PBMCs can be used directly.
4. Harvest cells, wash once with PBS, centrifuge at 300 × g for 5 minutes.
5. Resuspend in FACS buffer (PBS + 2% FBS + 0.05% sodium azide) at 1 × 10⁷ cells/mL.
6. Aliquot 100 μL (1 × 10⁶ cells) per well into a 96-well V-bottom plate.
For mouse samples: harvest splenocytes or lymph node cells, prepare single-cell suspension through a 70 μm cell strainer. For activated mouse T cells, stimulate with anti-CD3ε (clone 145-2C11, 1 μg/mL) + anti-CD28 (clone 37.51, 2 μg/mL) for 48 hours.
Important: CTLA-4 surface expression peaks at 48–72 hours post-activation and is rapidly internalized. Timing of staining is critical.
Viability Staining
Viability Staining
1. Centrifuge at 300 × g for 5 minutes, discard supernatant.
2. Resuspend in 100 μL PBS containing fixable viability dye per manufacturer's instructions.
3. Incubate at room temperature for 15 minutes, protected from light.
4. Wash once with FACS buffer.
Note: Use a FIXABLE viability dye (not PI or 7-AAD) since downstream intracellular staining requires fixation, which eliminates live/dead discrimination by membrane-impermeable dyes.
Fc Blocking
Fc Blocking
Human samples:
1. Resuspend cells in 50 μL FACS buffer containing human Fc block (5 μL per test).
2. Incubate at room temperature for 10 minutes.
3. Do NOT wash. Proceed directly to surface staining.
Mouse samples:
1. Resuspend cells in 50 μL FACS buffer containing anti-mouse CD16/CD32 Fc block.
- abinScience InVivoMAb Mouse Anti-Mouse CD16/CD32 (Iv0016) (Cat# MP551010): https://www.abinscience.com/product/40/4094.html
2. Incubate at 4°C for 10 minutes.
3. Do NOT wash.
Surface Staining
Surface Staining (Protocol A — for surface CTLA-4 on activated T cells)
This step detects CTLA-4 transiently expressed on the cell surface. Perform on ACTIVATED T cells only.
Recommended directly conjugated antibodies:
Human CTLA-4:
- Anti-Human CD152/CTLA4 Antibody (11.2.1), FITC (Cat# HB651217): https://www.abinscience.com/product/40/4923.html
- Anti-Human CD152/CTLA4 Antibody (11.2.1), PE (Cat# HB651227): https://www.abinscience.com/product/40/7944.html
- Anti-Human CD152/CTLA4 Antibody (11.2.1), APC (Cat# HB651237): https://www.abinscience.com/product/40/5930.html
Mouse CTLA-4:
- Anti-Mouse CD152/CTLA4 Antibody (9D9), PE (Cat# MB651227): https://www.abinscience.com/product/40/7948.html
- Anti-Mouse CD152/CTLA4 Antibody (9D9), APC (Cat# MB651237): https://www.abinscience.com/product/40/5934.html
- Anti-Mouse CD152/CTLA4 Antibody (9D9), FITC (Cat# MB651217): https://www.abinscience.com/product/40/4927.html
1. Add fluorochrome-conjugated anti-CTLA-4 antibody at recommended concentration per datasheet.
2. Co-stain with lineage markers: anti-CD3 (T cells), anti-CD4, anti-CD25 (for Treg identification).
3. Incubate at 4°C for 30 minutes, protected from light.
4. Wash twice with 200 μL FACS buffer, centrifuge at 300 × g for 5 minutes.
If surface-only detection is sufficient, proceed to Step 7 (Acquisition). For total CTLA-4 (surface + intracellular), continue to Step 5.
Fixation and Permeabilization
Fixation and Permeabilization (Protocol B — for intracellular CTLA-4)
This step is REQUIRED for detecting total CTLA-4, including the intracellular pool stored in vesicles. This is the recommended approach for Treg characterization (CD4+ CD25+ FoxP3+ CTLA-4+).
1. After surface staining (or directly after Fc block if skipping surface staining), fix cells in 200 μL fixation buffer (2–4% paraformaldehyde in PBS) at 4°C for 20 minutes.
2. Wash once with FACS buffer, centrifuge at 300 × g for 5 minutes.
3. Resuspend in 200 μL permeabilization buffer (0.1% saponin in FACS buffer, or commercial perm buffer).
4. Centrifuge and resuspend in 100 μL permeabilization buffer.
Note: For combined FoxP3 + CTLA-4 staining in Tregs, use a dedicated FoxP3/Transcription Factor Staining Buffer Set — the nuclear permeabilization step is also sufficient for CTLA-4 detection.
Important: PE and APC conjugates retain brightness after fixation/permeabilization. FITC is also compatible. PerCP may show reduced signal after some fixation protocols — test with your specific buffer set.
Intracellular CTLA-4 Staining
Intracellular CTLA-4 Staining
1. Add fluorochrome-conjugated anti-CTLA-4 antibody in permeabilization buffer at recommended concentration.
For intracellular staining, use a DIFFERENT fluorochrome from the one used for surface CTLA-4 (if performing combined surface + intracellular protocol to distinguish the two pools).
Recommended antibodies — same clones as Step 4, choose a different conjugate:
- Anti-Human CD152/CTLA4 (11.2.1), PE (Cat# HB651227): https://www.abinscience.com/product/40/7944.html
- Anti-Human CD152/CTLA4 (11.2.1), APC (Cat# HB651237): https://www.abinscience.com/product/40/5930.html
- Anti-Mouse CD152/CTLA4 (9D9), PE (Cat# MB651227): https://www.abinscience.com/product/40/7948.html
- Anti-Mouse CD152/CTLA4 (9D9), APC (Cat# MB651237): https://www.abinscience.com/product/40/5934.html
Unconjugated options (requires fluorochrome-conjugated secondary antibody):
- Anti-Human CD152/CTLA4 (11.2.1) (Cat# HB651207): https://www.abinscience.com/product/40/3655.html
- Research Grade Ipilimumab (Cat# HB651016): https://www.abinscience.com/product/71/1369.html
2. Incubate at room temperature for 30 minutes in the dark.
3. Wash twice with permeabilization buffer (NOT regular FACS buffer — saponin-based permeabilization is reversible).
4. Resuspend in 200 μL FACS buffer for acquisition.
Acquisition and Analysis
Acquisition and Analysis
1. Acquire ≥10,000 events in the CD3+ T cell gate (or ≥50,000 total events for rare Treg populations).
2. Gating strategy:
- FSC/SSC → singlets (FSC-A vs FSC-H) → viable cells → CD3+ → CD4+ or CD8+
- For Tregs: CD3+ → CD4+ → CD25 high → FoxP3+ → CTLA-4+
- For activated T cells (surface CTLA-4): CD3+ → CD4+ → CTLA-4+
3. Controls:
- Isotype control or FMO (fluorescence minus one) control to define CTLA-4 positive gate
- Unstimulated T cells as biological negative control for surface CTLA-4
- Stimulated T cells as positive control for surface expression
4. Expected results:
- Surface CTLA-4: <5% on resting T cells; 20–60% on CD4+ T cells after 48–72 h anti-CD3/CD28 stimulation
- Intracellular CTLA-4: 80–95% of CD4+ CD25high FoxP3+ Tregs; 10–30% of resting conventional CD4+ T cells
- Mouse: similar pattern; 9D9 clone detects both surface and intracellular CTLA-4
5. For in vivo CTLA-4 blocking studies, abinScience also offers:
- InVivo Plus Anti-Mouse CD152/CTLA4 (9D9) (Cat# MB651209): https://www.abinscience.com/product/68/28328.html