Journal: MethodsX

Article Title: Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol

doi: 10.1016/j.mex.2026.103869

Figure Lengend Snippet: Representative leukocyte-gated histograms illustrating antibody titration for feline leukocyte immunophenotyping. All histograms display singlet leukocytes, defined by FSC-A versus SSC-A morphological gating, followed by FSC—H versus FSC-A singlet discrimination. Titration was performed for CD18, CD21, CD45R, CD4, CD5 and CD8 monoclonal antibodies using three antibody volumes: 10 µL (1:10 dilution), 5.0 µL (1:20 dilution), 3.0 µL (1:33 dilution) and 1.5 µL (1:66 dilution). Minimal working volumes were selected based on optimal signal-to-noise ratios.

Article Snippet: Step 2 – Leukocytes extracellular staining Materials • EDTA Whole blood sample ± Transfix® • 200 μl pipettes • 100 μl pipettes • 10 μl pipettes • Flow cytometry tubes • Permanent marker • Cytometer tube rack Reagents • Monoclonal antibodies: ○ CD5 Anti-cat – clone FE1.1B11 (BIO-RAD®) ○ CD4 Anti-cat – clone vpg34 (BIO-RAD®) ○ CD8 Anti-cat alpha/beta purified – clone vpg9 (BIO-RAD®) ○ Rat Anti-Mouse IgG1 – clone X56 (BIO-RAD®) ○ CD18 Mouse Anti-Dog – clone CA1.4E9 (BIO-RAD®) ○ CD21 Mouse Anti-Dog – clone CA2.1D6 (BIO-RAD®) ○ CD45R Rat Anti-Mouse – clone RA3–6B2 (BIO-RAD®) • 10x Red blood cells (RBC) lysis buffer solution (BD FACSTM lysing solution) • PBS 1% solution Equipment • Countess TM 3 (Thermo Fisher Scientific, USA) • Freezer • Dark incubation chamber • Timer • Vortex (MX-S®, China) • Centrifuge (model 5810R, Eppendorf®, Germany) • Flow cytometry analyser BD FACSCanto II (Becton Dickinson (BD), San Jose, USA) Methods 1.

Techniques: Titration, Bioprocessing