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Applications & Panels

Staining protocols and validated panel resources for the UTSW Flow & Mass Cytometry Core

The Flow & Mass Cytometry Core provides validated staining protocols and curated panel resources to help investigators design reproducible, high-quality flow cytometry experiments. This guide describes three Core staining protocols covering standard surface staining, intracellular/nuclear antigen detection, and cell sorting sample preparation, as well as an example validated panel for intestinal macrophage identification.

Investigators are encouraged to consult Core staff before beginning new experiments, particularly for multi-color panel design, cell sorting, and tissue-specific isolation workflows.

Do you have questions?

Reach out to Core staff via FlowCytometry@UTSouthwestern.edu or contact Dr. Dende through Chaitanya.Dende@UTSouthwestern.edu or by phone at (214) 648-7332.

Staining Protocol Library

The following Core protocols provide step-by-step instructions optimized for the reagents and instruments available at UTSW.

Analysis

General Flow Cytometry Staining Guidelines

Standard workflow for surface marker staining (analysis runs)

  • Sample preparation: single-cell suspension, RBC lysis, resuspension in FACS buffer (PBS + 3% FBS + 0.5 mM EDTA)
  • Fc receptor blocking prior to antibody addition (10 min, separate step for myeloid populations)
  • Surface antibody staining: 15–20 min at 4°C, protected from light
  • Optional fixation in ~1% paraformaldehyde for delayed acquisition
  • Compatible with directly conjugated, biotin–streptavidin, and primary/secondary antibody approaches

Staining Guidelines for Flow Cytometry Analysis

This section provides general staining guidelines for flow cytometry analysis based on established Core protocols. These guidelines are intended to promote data quality and reproducibility. Investigators are encouraged to consult Core staff for experiment-specific recommendations.

  • Step 1: Sample Preparation and Blocking
    1. Prepare single-cell suspensions from tissues, blood, or cultured cells.
    2. Approximately 1 × 105 -1 × 10⁶ cells per sample is generally sufficient for staining.
    3. Red blood cells should be lysed prior to staining when working with blood or highly vascular tissues.
    4. Wash cells and resuspend in FACS buffer (PBS supplemented with 3% FBS, 0.5 mM EDTA, and sodium azide).
    5. Perform Fc receptor blocking prior to antibody staining to minimize nonspecific binding.
    6. For myeloid or Fc receptor-rich populations, Fc block should be performed as a separate incubation step (10 minutes) before antibody addition.
  • Step 2: Antibody Staining
    1. Flow cytometry staining may involve directly conjugated antibodies, biotin–streptavidin systems, or primary and secondary antibody approaches.
    2. Directly conjugated antibodies are recommended for one-step surface staining when possible.
    3. For multi-step staining, incubate biotinylated or primary antibodies first, followed by streptavidin or secondary antibody incubation.
    4. Surface antibody staining is typically performed at 4°C and protected from light.
    5. Incubation times of approximately 15 to 20 minutes are generally sufficient.
  • Step 3: Fixation and Storage
    1. If data acquisition will be delayed, samples may be fixed using FACS buffer containing paraformaldehyde to a final concentration of approximately 1%.
    2. Fixed samples should be protected from light and stored at 4°C until acquisition.

Sorting

Cell Sorting Staining & Sample Preparation Guidelines

Workflow for live cell sorting (viability and purity optimized)

  • High viability required (>90%) prior to sorting for optimal purity and recovery
  • Filter samples through 40 µm nylon mesh immediately before loading on the sorter
  • Directly conjugated antibodies strongly recommended; multi-step staining requires advance coordination
  • Include a fixable viability dye to exclude dead cells during sorting
  • Collect sorted cells into 3% FBS/BSA-supplemented buffer or HEPES-buffered media
  • Fixation and permeabilization are generally incompatible with live cell sorting (discuss with Core staff in advance)

Staining and Sample Preparation Guidelines for Cell Sorting

This section provides general staining and sample preparation guidelines for cell sorting based on established Core protocols. Because sorting performance and post-sort viability are highly dependent on sample quality, investigators are encouraged to consult Core staff prior to sorting for experiment-specific recommendations.

  • Step 1: Sample Preparation and Blocking
    1. Prepare high-quality single-cell suspensions from tissues, blood, or cultured cells.
    2. Cell viability should be high (>90%) prior to sorting to ensure optimal purity and recovery.
    3. Red blood cells should be lysed prior to staining when working with blood or highly vascular tissues.
    4. Suspend cells in a low-protein sorting buffer (e.g., PBS with 3% FBS and 0.5 mM EDTA) to maintain viability and reduce aggregation.
    5. Filter samples through a 40 µm nylon mesh immediately prior to sorting to prevent nozzle clogs.
    6. Perform Fc receptor blocking prior to antibody staining to minimize nonspecific binding.
    7. For myeloid or Fc receptor-rich populations, Fc block should be performed as a separate incubation step (approximately 10 minutes) before antibody addition.
  • Step 2: Antibody Staining
    1. Staining panels for cell sorting should prioritize marker resolution, cell viability, and minimal nonspecific binding.
    2. Directly conjugated antibodies are strongly recommended for sorting experiments to minimize staining steps and sort time.
    3. Multi-step staining strategies (e.g., biotin–streptavidin or primary/secondary antibodies) should be used only when necessary and discussed with Core staff in advance.
    4. Include a viability dye whenever possible to exclude dead cells during sorting.
    5. Surface antibody staining is typically performed at 4°C and protected from light.
    6. Incubation times of approximately 15 to 20 minutes are generally sufficient for surface staining.
  • Step 3: Post-Stain Handling and Collection
    1. After staining, wash samples thoroughly to remove unbound antibodies.
    2. Filter samples again immediately prior to loading on the sorter if aggregates are present.
    3. Keep samples protected from light and at appropriate temperatures until sorting begins.
    4. Fixation and permeabilization are generally incompatible with live cell sorting and should be avoided unless discussed with Core staff in advance.
    5. Sorted cells should be collected into tubes or plates containing 3% FBS/BSA-supplemented buffer or media to improve post-sort viability.
    6. If sorting into media, ensure your cell preferred media or the media is HEPES-buffered to maintain pH under ambient conditions.

Intracellular

Nuclear Antigen/Foxp3 Staining Guidelines

Workflow for transcription factor and nuclear antigen detection

  • Viability staining first (fixable dye, e.g., Ghost 710), then complete all surface staining before fixation
  • Fixation and permeabilization using a transcription factor–compatible commercial kit (e.g., Foxp3 Fix/Perm)
  • Nuclear antibody incubation in 1× Permeabilization Buffer: 30–45 min at room temperature
  • Standard cytoplasmic cytokine permeabilization buffers are NOT compatible (use transcription factor kit)
  • Verify antibody clone compatibility with transcription factor fixation reagents before use
  • Include FMO controls; optimization may be required across species, tissues, and targets

Nuclear Antigen (Foxp3) Staining Guidelines

These guidelines describe a standard workflow for staining nuclear antigens (e.g., Foxp3) using commercial fixation and permeabilization kits designed for transcription factor detection. Nuclear antigen staining requires specialized fixation and permeabilization conditions distinct from cytoplasmic cytokine staining. Investigators are encouraged to consult Flow Cytometry Core staff when adapting this workflow for additional nuclear targets.

  • Overview of Workflow
    1. Cell preparation and viability staining
    2. Surface marker staining
    3. Fixation and permeabilization using a transcription factor–compatible kit
    4. Nuclear antigen antibody staining
    5. Data acquisition
  • Step 1: Cell Preparation and Viability Staining
    1. Prepare a single-cell suspension according to standard flow cytometry sample preparation guidelines.
    2. Distribute cells into a 96-well round-bottom plate or appropriate tube format.
    3. Reserve a portion of cells for compensation and control samples.
    4. Wash cells with ice-cold PBS.
    5. Stain cells with a fixable viability dye (e.g., Ghost 710) diluted 1:500 in PBS.
    6. Incubate for 20 minutes at room temperature, protected from light.
    7. Wash cells with FACS buffer (PBS supplemented with 3% FBS or BSA and 0.5 mM EDTA).
  • Step 2: Fc Blocking and Surface Marker Staining
    1. Block Fc receptors using Fc block diluted 1:1000 in FACS buffer.
    2. Incubate for 10 minutes at 4°C.
    3. Without washing, add fluorochrome-conjugated surface antibodies diluted in FACS buffer.
    4. Incubate for 20 minutes at 4°C, protected from light.
    5. Wash cells 2 to 3 times with FACS buffer to remove unbound antibodies.
    6. Complete all surface staining prior to fixation, as transcription factor fixation can alter surface epitopes.
  • Step 3: Fixation and Permeabilization (Foxp3 Kit)
    1. Fully resuspend cell pellets and add Foxp3 Fixation/Permeabilization Working Solution (typically 200 µL per sample).
    2. Ensure complete resuspension by gentle pipetting.
    3. Incubate for 30–60 minutes at room temperature, protected from light.
    4. Mouse samples may be incubated for up to 18 hours at 2–8°C if required.
    5. Wash cells with 2× Permeabilization Buffer and repeat wash once.
  • Step 4: Nuclear Antigen Staining
    1. Resuspend cells in 1× Permeabilization Buffer (final volume approximately 100 µL).
    2. Optional: Block with 2% normal mouse or rat serum for 15 minutes at room temperature.
    3. Without washing, add directly conjugated antibodies against Foxp3 or other nuclear antigens.
    4. Incubate for at least 30 to 45 minutes at room temperature, protected from light.
    5. Wash cells twice with 1× Permeabilization Buffer.
  • Step 5: Final Resuspension and Data Acquisition
    1. Resuspend cells in Flow Cytometry Staining Buffer or FACS buffer.
    2. Samples may be analyzed immediately or stored at 4°C, protected from light, for short periods.
    3. Acquire data on the flow cytometer using appropriate compensation and controls.
  • Important Notes and Recommendations
    1. Use commercial transcription factor staining kits for nuclear antigen detection, as standard cytokine permeabilization buffers are not compatible.
    2. Verify antibody compatibility with transcription factor fixation reagents prior to use.
    3. Include appropriate controls, such as unstained, single-stain, and fluorescence-minus-one (FMO) controls.
    4. Nuclear antigen staining may require optimization depending on species, tissue source, and target abundance.

Which protocol do I need?

Analyzing surface markers onlySorting live cellsStaining Foxp3, Ki67, or other nuclear targets
General staining guidelines Sorting staining and sample prep guidelines Nuclear Antigen/Foxp3 staining guidelines

Panel Library

The Core maintains a library of validated, publication-ready panels optimized for specific tissues and cell populations. Panels are tested and annotated with antibody clones, fluorochrome assignments, laser/detector configurations, and sequential gating strategies. Investigators can use these panels as-is or adapt them with guidance from Core staff.

Featured Panel

Colonic/Small Intestinal LPL — Myeloid Macrophage Identification Panel

  • Tissue: Colon / Small Intestine (Lamina Propria)
  • Species: Mouse
  • Colors: 4 (screening panel)
  • Instrument: Cytek Aurora or BD Analyzer

A 4-color screening panel for identifying lamina propria macrophages by sequential gating through scatter, singlet, viability/CD45, and macrophage (CD64⁺ CD11b⁺) gates. Includes Ghost Dye 710 viability, CD45-BUV395, CD64-StarBright Blue 700, and CD11b-FITC. Companion isolation protocol available.

Key populations identified: Tissue-resident macrophages (CD64⁺ CD11b⁺), monocytes, lymphocytes, stromal cells

See the Featured Panel Protocol Reference Sheet

Additional Panels & Custom Panel Design

Additional validated panels are available for lymphocyte immunophenotyping, DC subsets, and other myeloid populations. For custom panel design or tissue-specific isolation advice, FlowCytometry@UTSouthwestern.edu Core staff to schedule a consultation.
 

Getting Help

Core staff and Dr. Dende are available to assist with all aspects of experiment design and execution. We recommend a pre-experiment consultation for:

  • New panel design or adaptation of an existing Core panel
  • Cell sorting experiments (required prior coordination for all sorts)
  • Tissue-specific isolation protocols (e.g., lamina propria, tumor microenvironment, lymph node)
  • Spectral unmixing, high-parameter panels, or mass cytometry (CyTOF) experiments
  • Troubleshooting data quality, compensation, or gating questions
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