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Technical Specification: Flow Cytometry Reagents

1. Product Range

Solarbio flow cytometry reagents cover fluorophore-conjugated antibodies for immunophenotyping, apoptosis detection reagents, cell preparation buffers, and DNA content analysis reagents. All antibodies are titrated and validated for flow cytometry applications.

Product SKU Range Available Conjugations Clone Reactivity
CD3 Antibody (Flow) F1001–F1010 FITC, PE, APC, PerCP-Cy5.5, PE-Cy7 UCHT1 (human), 17A2 (mouse) Human, mouse
CD4 Antibody (Flow) F1020–F1029 FITC, PE, APC, BV421, PerCP RPA-T4 (human), GK1.5 (mouse) Human, mouse
CD8 Antibody (Flow) F1040–F1049 FITC, PE, APC, BV421, PE-Cy7 RPA-T8 (human), 53-6.7 (mouse) Human, mouse
CD19 Antibody (Flow) F1060–F1069 FITC, PE, APC, PerCP-Cy5.5 HIB19 (human), 6D5 (mouse) Human, mouse
CD45 Antibody (Flow) F1080–F1089 FITC, PE, APC, PerCP, BV421 HI30 (human), 30-F11 (mouse) Human, mouse
Annexin V-FITC/PI CA1020 FITC + PI — All species
7-AAD Viability Staining Solution P8110 — — All species
RNase A (DNase-free) R1030 — — For cell cycle analysis

2. Fluorophore Selection and Spectral Characteristics

Fluorophore Ex Max (nm) Em Max (nm) Laser Line Typical Detector Relative Brightness Photostability
FITC 495 520 488 nm FL1 (530/30) Medium Moderate
PE (R-Phycoerythrin) 565 576 488 nm FL2 (585/40) Very high High
PerCP 479 675 488 nm FL3 (670/LP) Medium Low
PerCP-Cy5.5 482 695 488 nm FL3 (695/40) High Moderate
PE-Cy7 565 774 488 nm FL4 (780/60) High Low (use with care)
APC 650 660 633/640 nm FL5 (660/20) High High
APC-Cy7 650 775 633/640 nm FL6 (780/60) High Low
BV421 (Brilliant Violet 421) 405 421 405 nm FL7 (450/50) Very high Moderate
BV510 405 510 405 nm FL8 (525/50) High Moderate
PI (Propidium Iodide) 535 615 488 nm FL3 (670/LP) Medium High
7-AAD 546 647 488 nm FL3 (670/LP) Medium High

3.1 Human T-cell Subtyping Panel (4-Color)

Marker Fluorophore Function Expected Population
CD45 PerCP Pan-leukocyte gate All leukocytes
CD3 FITC T-cell marker CD45⁺CD3⁺: 55–85% of lymphocytes
CD4 PE Helper T-cells CD3⁺CD4⁺: 30–60% of T cells
CD8 APC Cytotoxic T-cells CD3⁺CD8⁺: 15–40% of T cells

3.2 Human B-cell Identification (3-Color)

Marker Fluorophore Function Expected Population
CD45 PerCP Pan-leukocyte gate All leukocytes
CD19 FITC B-cell marker CD45⁺CD19⁺: 5–15% of lymphocytes
CD20 PE Mature B-cell marker CD19⁺CD20⁺: >90% of B cells

3.3 Mouse Immunophenotyping (6-Color)

Marker Fluorophore Laser Cell Type
CD45 BV510 405 nm All leukocytes
CD3 FITC 488 nm T cells
CD4 PE 488 nm Helper T cells
CD8 APC 633 nm Cytotoxic T cells
CD19 PerCP-Cy5.5 488 nm B cells
NK1.1 PE-Cy7 488 nm NK cells

3.4 Apoptosis Detection

Marker Fluorophore Detection Interpretation
Annexin V FITC FL1 (530/30) Phosphatidylserine externalization
PI PI FL3 (670/LP) Membrane integrity
7-AAD 7-AAD FL3 (670/LP) Alternative to PI for live/dead gating

3.5 Cell Cycle Analysis (PI DNA Staining)

Parameter Specification
Cell number 1×10⁶ cells per sample
Fixation 70% ethanol at -20°C, ≥2 h (up to 1 week)
RNase A 100 μg/mL, 30 min at 37°C
PI staining 50 μg/mL, 30 min at RT (dark)
Acquisition mode Linear (not log) for PI-Area; doublet discrimination with PI-W vs. PI-A

4. Buffer Specifications

Buffer Content pH Storage Recommended Use
10× Flow Cytometry Staining Buffer PBS, 2% BSA, 0.1% NaN₃ 7.4 2–8°C, 12 months Antibody dilution, cell resuspension, washing
1× RBC Lysis Buffer 0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA 7.2 RT, 12 months Whole blood RBC lysis (10 min at RT)
Fixation Buffer 4% paraformaldehyde in PBS 7.4 2–8°C, 6 months (dark) Cell fixation post-staining
Permeabilization Buffer 0.1% saponin + 1% BSA in PBS 7.4 2–8°C, 12 months Intracellular staining; use with 0.3% saponin
Cell Cycle Staining Buffer PBS + 0.1% Triton X-100 + RNase A + PI 7.4 Prepare fresh Cell cycle analysis

5. Antibody Titration Protocol

Accurate antibody titration is essential for optimal signal-to-noise ratio in flow cytometry. The recommended titration range is 1:25 to 1:800 (or 0.25–8 μg/mL).

Dilution Stain Index (Median Positive — Median Negative) / (2× rSD of Negative) Recommendation
1:25 25.3 Slightly above saturation; high reagent consumption
1:50 28.7 Optimal (highest stain index)
1:100 27.1 Good; reduced reagent consumption
1:200 21.5 Acceptable for highly expressed antigens
1:400 14.2 Below optimal

For a new antibody, titrate with 1×10⁵ positive cells per condition. The optimal dilution is the lowest concentration yielding maximal stain index.

6. Compensation and Controls

Control Type Purpose Composition
Unstained control Autofluorescence baseline Cells only, no antibodies
Single-color compensation controls Spectral overlap compensation One fluorophore per tube; use compensation beads for each fluorophore
FMO (Fluorescence Minus One) controls Gate boundary setting All fluorophores except the one being gated
Isotype control Confirm specific binding Same concentration of isotype-matched non-specific antibody

7. Troubleshooting

Issue Possible Cause Solution
Low cell yield Excessive washing/centrifugation Reduce washing steps; use 300 × g, 5 min; resuspend gently
High debris (forward scatter low) Dead cells; sample stored too long Increase live/dead gate; add viability dye; use fresh samples
Doublet populations Cell clumping; insufficient disaggregation Filter through 40 μm mesh; reduce cell density; add DNase I
Poor separation (positive vs. negative) Antibody under-titrated; target low expression Titrate antibody; use brighter fluorophore; increase cell number
High background (all cells positive) Non-specific binding; FC receptors Add Fc block (2.4G2 for mouse; human IgG for human); include isotype control
Compensation errors (all in all channels) Insufficient compensation controls; spectral overlap Use single-color controls for each fluorophore; use compensation matrix
Dim signal despite high expression Fluorophore degradation; photobleaching Store antibodies at 4°C in dark; prepare fresh staining solution; add fixative after staining

8. Cross-References

For product procurement: solarbio.store