Flow Cytometry — Armin Bayati, PhD

01

Populations

Cell Population Frequencies

Panel 1 quantifies CD4+ T cells, CD8+ T cells, B cells, Tregs, and Ki67+ proliferating subsets across WT and LRRK2 genotypes treated with PBS, PFF, or compound X1. Bar charts show group means with SEM error bars and individual animal data points overlaid.

6

Treatment groups (WT/LRRK2 × 3 treatments)

7

Cell populations profiled per panel

2

Tissue types (LN & Spleen)

02

Histograms

Fluorescence Distributions

Arcsinh-transformed fluorescence histograms for Panel 3 myeloid markers — Ki67, CD11c, CD11b, F4/80, Ly6G, Ly6C — with all specimens overlaid and the unstained control shown as a dashed black line. These distributions reveal marker expression levels and the separation between positive and negative populations.

03

Gating

FSC / SSC Scatter

Forward scatter (FSC-A) vs side scatter (SSC-A) dot plots are the first step in the gating hierarchy, used to identify the main cell population and exclude debris and doublets before downstream immune phenotyping.

04

Protocols

Flow Cytometry Protocols & Equipment

Detailed protocols for surface and intracellular flow cytometry staining, as well as fluorescence-activated cell sorting (FACS) on the BD FACSAria III. All analytical acquisition is performed on the BD LSRFortessa (4-laser, up to 18-parameter configuration) or the Cytek Aurora spectral cytometer (5-laser, full-spectrum). Sorting is performed on the BD FACSAria III equipped with 4 lasers (405 nm, 488 nm, 561 nm, 640 nm), 100 µm nozzle, and 4-way sorting capability.

BD LSRFortessa

4-laser analytical cytometer (405/488/561/640 nm) — up to 18 parameters

BD FACSAria III

4-laser high-speed sorter — 4-way sorting, 100 µm nozzle, biosafety containment

Cytek Aurora

5-laser spectral cytometer — full-spectrum unmixing for high-parameter panels

FlowJo v10

Analysis software — compensation, gating, t-SNE/UMAP dimensionality reduction

Surface Staining Protocol (Standard Flow Cytometry)

This is my standard multi-color surface staining protocol used for immunophenotyping lymphocyte and myeloid populations from mouse tissue (spleen, lymph node) or human PBMCs. All steps performed on ice or at 4 °C unless otherwise noted.

Protocol — Surface Staining for Flow Cytometry

1. Tissue Dissociation & Single-Cell Suspension

Harvest spleen or lymph nodes into ice-cold RPMI + 2% FBS. Mechanically dissociate tissue through a 70 µm cell strainer using a syringe plunger. Rinse strainer with 5 mL FACS buffer (PBS + 2% FBS + 2 mM EDTA). For spleen: lyse red blood cells with ACK lysis buffer (2 min, RT), then quench with 10 mL FACS buffer. Centrifuge 400 × g, 5 min, 4 °C. Resuspend in FACS buffer and count viable cells using trypan blue exclusion.

2. Viability Staining

Aliquot 1 × 10⁶ cells per condition into 96-well V-bottom plate. Wash 1× with PBS (no protein). Resuspend in 100 µL PBS containing Zombie Aqua™ fixable viability dye (1:500 dilution). Incubate 15 min, RT, protected from light. Wash 2× with FACS buffer.

3. Fc Receptor Blockade

Resuspend cell pellet in 50 µL FACS buffer containing anti-CD16/CD32 Fc block (clone 2.4G2, 1:100) for mouse cells, or Human TruStain FcX™ (1:20) for human PBMCs. Incubate 10 min on ice. Do not wash — proceed directly to antibody staining.

4. Surface Antibody Cocktail

Prepare antibody master mix at pre-titrated concentrations in FACS buffer. Add 50 µL antibody cocktail directly to cells (final volume 100 µL). Typical Panel 1 (Lymphoid): CD45-BV785, CD3-BV421, CD4-FITC, CD8-PE-Cy7, CD19-APC, NK1.1-PE, CD25-BV605, CD44-APC-Cy7, CD62L-PerCP-Cy5.5. Typical Panel 3 (Myeloid): CD45-BV785, CD11b-BV421, CD11c-PE, F4/80-APC, Ly6G-FITC, Ly6C-PerCP-Cy5.5, MHC-II-BV605. Incubate 30 min on ice, protected from light.

5. Wash & Fix

Wash 2× with 200 µL FACS buffer (400 × g, 5 min, 4 °C). For immediate acquisition: resuspend in 200 µL FACS buffer and proceed to cytometer. For delayed acquisition (up to 72 h): fix in 100 µL 1% PFA in PBS for 15 min at 4 °C, wash 1×, resuspend in 200 µL FACS buffer, store at 4 °C protected from light.

6. Acquisition on BD LSRFortessa

Run CS&T beads daily for cytometer QC. Set voltages using unstained and single-stain compensation controls (CompBeads or single-stained cells). Acquire compensation matrix in BD FACSDiva™ software. Record ≥ 50,000 live singlet events per sample at medium flow rate (~1,000–3,000 events/sec). Export .fcs files for analysis in FlowJo v10.

7. Gating Strategy

Standard hierarchy: FSC-A vs SSC-A (exclude debris) → FSC-H vs FSC-A (singlet gate) → Viability dye⁻ (live cells) → CD45+ (leukocyte gate) → lineage markers for subset identification. Apply fluorescence-minus-one (FMO) controls to set positive/negative gates for each marker. Export population statistics and overlays for publication-quality figures.

Intracellular Staining Protocol (Ki67 / Cytokines / FoxP3)

Extended protocol for intracellular targets requiring fixation and permeabilization. Used for Ki67 proliferation analysis, FoxP3 Treg identification, and intracellular cytokine staining (ICS) following PMA/ionomycin stimulation with brefeldin A/monensin.

Protocol — Intracellular Staining (Fix/Perm)

1. Surface Staining (Steps 1–5 above)

Complete surface staining and viability staining as described above. Do not fix with PFA if using the eBioscience FoxP3/Transcription Factor Staining Buffer Set.

2. Fixation & Permeabilization

For nuclear targets (Ki67, FoxP3): Use eBioscience FoxP3/Transcription Factor Staining Buffer Set. Add 200 µL Fix/Perm working solution, vortex immediately, incubate 30–60 min at 4 °C. Wash 2× with 1× Permeabilization Buffer.
For cytoplasmic targets (IFN-γ, TNF-α, IL-2): Use BD Cytofix/Cytoperm™. Add 100 µL Cytofix/Cytoperm solution, incubate 20 min at 4 °C. Wash 2× with 1× BD Perm/Wash™ Buffer.

3. Intracellular Antibody Staining

Resuspend permeabilized cells in 100 µL Perm Buffer containing intracellular antibodies at pre-titrated dilutions (e.g., Ki67-PE 1:200, FoxP3-APC 1:100, IFN-γ-FITC 1:100). Incubate 30 min at 4 °C, protected from light. Wash 2× with Perm Buffer. Resuspend in 200 µL FACS buffer for acquisition.

4. Acquisition & Analysis

Acquire on BD LSRFortessa as above. Use isotype controls and FMO controls to gate intracellular markers, as fixation/perm can increase background fluorescence. For Ki67: gate Ki67+ vs Ki67− within each T-cell subset. For FoxP3: gate CD4+CD25+FoxP3+ Tregs. For ICS: report % cytokine-positive within CD4+ or CD8+ gates after subtracting unstimulated control values.

BD FACSAria III — FACS Sorting Protocol

High-purity cell sorting protocol on the BD FACSAria III for isolating specific immune populations for downstream functional assays, RNA extraction, or single-cell genomics. I routinely sort rare populations (Tregs, antigen-specific T cells, tissue-resident macrophages) to > 95% purity with > 85% post-sort viability.

Protocol — BD FACSAria III Cell Sorting

1. Instrument Setup & QC

Power on BD FACSAria III and allow 15 min laser warm-up (405 nm violet, 488 nm blue, 561 nm yellow-green, 640 nm red). Run CS&T beads for automated QC and baseline performance tracking. Install 100 µm nozzle (standard for immune cells; use 85 µm for smaller cells or 130 µm for large/fragile cells). Set sheath pressure to 20 psi (100 µm) or 45 psi (70 µm). Run Accudrop beads to optimize drop delay and verify sort accuracy into a test tube.

2. Sample Preparation for Sorting

Prepare single-cell suspension and stain as per surface staining protocol above. Critical differences for sorting: resuspend at 10–20 × 10⁶ cells/mL in sort buffer (PBS + 1% BSA + 25 mM HEPES + 1 mM EDTA, sterile-filtered, 0.22 µm). Filter through 35 µm nylon mesh cap (BD Falcon™ FACS tube) immediately before sorting to prevent clogging. Keep cells on ice and protected from light.

3. Sort Configuration

Define sort gates in BD FACSDiva™ using the same gating hierarchy as analytical flow (FSC/SSC → singlets → live → CD45+ → lineage gates). Select sort mode based on application: Purity mode (> 99% purity, lower yield — for genomics/proteomics), 4-Way Purity (sorting up to 4 populations simultaneously), or Single-Cell mode (index sorting into 96/384-well plates for scRNA-seq or clonal expansion). Set sort mask: 1-2-0 (yield-purity-phase) for standard sorts, 0-32-0 for maximum purity.

4. Collection & Post-Sort Processing

Collect sorted cells into 15 mL tubes pre-coated with FBS (add 1 mL FBS, rotate to coat walls, then add 2 mL complete medium). For plate sorts: pre-fill wells with lysis buffer (scRNA-seq) or complete medium (functional assays). Sort at ≤ 5,000 events/sec for optimal purity and viability. Monitor sort efficiency and abort rate during the run; acceptable abort rate < 20%.

5. Post-Sort Purity Check

Re-run a small aliquot (~5,000 cells) of each sorted population on the FACSAria to verify sort purity ≥ 95%. Record post-sort purity and total cell yield. For viability-sensitive applications, perform trypan blue count or Annexin V/PI staining on a post-sort aliquot to confirm viability > 85%.

6. Downstream Applications

Sorted populations are used for: RNA extraction (RNeasy Micro for < 100K cells, RNeasy Mini for > 100K), scRNA-seq (10x Genomics Chromium), functional co-culture assays (e.g., sorted Tregs added back to effector T cells for suppression assays), Western blot / proteomics (≥ 500K cells per condition), or in vitro expansion (sorted T-cell subsets with anti-CD3/CD28 Dynabeads).

Equipment & Software Summary

Instrument	Application	Configuration
BD LSRFortessa	Analytical immunophenotyping	4 lasers, 18 detectors, HTS plate reader
BD FACSAria III	High-speed cell sorting	4 lasers, 4-way sort, 70/85/100 µm nozzles, ACDU
Cytek Aurora	Spectral flow cytometry	5 lasers, full-spectrum unmixing, 64-channel
BD FACSDiva™	Acquisition & instrument control	Compensation, gating, sort logic, CS&T QC
FlowJo v10	Post-acquisition analysis	Compensation, t-SNE/UMAP, batch analysis, plugins
SpectroFlo®	Cytek spectral unmixing	Reference controls, autofluorescence extraction

05

Methods

Detailed Methodology & Techniques

Comprehensive descriptions of key experimental techniques, assay platforms, and analytical methods referenced throughout this page.

Myeloid Panels

I design and run myeloid-focused flow cytometry panels for characterizing monocytes, macrophages, dendritic cells, and microglia in blood, tissue, and iPSC-derived models. Panels include CD14, CD16, CD11b, CD11c, CD68, CD163, CD206, HLA-DR, CX3CR1 with gating strategies to distinguish classical, intermediate, and non-classical monocyte subsets. These panels are critical for neuroinflammation studies and immuno-oncology characterization.

Fig. — Myeloid Subset Distribution

Relative frequencies of monocyte/macrophage subsets in PBMC vs brain tissue

12-color flow panel. BD LSRFortessa. n=6 donors.

Ki67 Proliferation

I quantify cellular proliferation using intracellular Ki67 staining combined with DNA content (DAPI/PI) for simultaneous proliferation and cell cycle analysis. This approach identifies cells in G0 (Ki67−/2N), G1 (Ki67+/2N), S (Ki67+/>2N), and G2/M (Ki67+/4N) phases. Applied to T-cell expansion post-stimulation, tumor cell proliferation under drug treatment, and iPSC-CM cell cycle exit during maturation.

Fig. — Ki67+ Proliferation Index

Ki67+ fraction across T-cell expansion timepoints post anti-CD3/CD28 stimulation

Intracellular Ki67 + DAPI. BD LSRFortessa. n=4 donors.

Lymph Node & Spleen Analysis

I process and analyze lymph node and spleen tissue from mouse models for comprehensive immune profiling. Tissue is mechanically dissociated, filtered, and stained with multi-parameter panels covering T cells, B cells, NK cells, dendritic cells, macrophages, and germinal center populations. These panels characterize immune responses in LRRK2 mouse models of Parkinson's disease and in tumor-bearing mice for immuno-oncology studies.

Fig. — Lymph Node Immune Composition

Major immune cell populations in WT vs LRRK2-G2019S mouse lymph nodes

16-color spectral flow. Cytek Aurora. n=8 mice per genotype.

LRRK2 Immune Profiling

I perform comprehensive immune profiling of LRRK2 mutant mice (G2019S knock-in) compared to wild-type controls. LRRK2 is highly expressed in immune cells and its gain-of-function mutation alters immune cell function. I characterize T-cell activation, myeloid polarization, cytokine secretion profiles, and tissue-resident immune populations across spleen, lymph nodes, and brain. These studies connect peripheral immune dysregulation to neuroinflammation in Parkinson's disease models.

Fig. — LRRK2 vs WT: Immune Cell Frequencies

Splenic immune cell populations in LRRK2-G2019S vs wild-type mice

Spectral flow cytometry. Cytek Aurora. n=8 per genotype. *p<0.05, **p<0.01.

Multiparameter Immune Profiling

Cell Population Frequencies

Fluorescence Distributions

FSC / SSC Scatter

Flow Cytometry Protocols & Equipment

Detailed Methodology & Techniques