Flow cytometry reagent and antibody

Human test subject

Buffy-coated blood (Healthy donor, n = 1) was obtained from the ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy.

PBMC isolation

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque (Sigma-Aldrich) following the standard protocol.

Plating and culture condition

After isolation, PBMCs were plated in a 96-well plate at 5×10⁵ cells per well and cultured at 37 °C, 5% CO₂ in either: RPMI-1640 + 10% FBS (baseline control), or RPMI-1640 + 10% FBS supplemented with 20% cell-free supernatant (CFS) from Intestinibacter bartlettii.

Rationale for conditioning

Some readouts of interest are stimulus dependent, IFN-γ (intracellular cytokine) and activation/exhaustion-related markers (CD69, PD-L1) change primarily after an external stimulus. To ensure that antibody performance was assessed across both negative and positive expression states, cells were conditioned with either RPMI or I. bartlettii CFS to model biological complexity and then subjected to functional stimulation. This approach maximizes the chance of detecting differences in marker expression, allows for direct assessment of antibody sensitivity.

Flow cytometry

Cells were stained with labeled antibodies diluted in PBS for 20 min at 4°C. For intracellular cytokine labelling cells were incubated for 3 h at 37°C in RPMI-1640 + 10% FBS with PMA (50 ng/ml, Merck), Ionomycin (1 μg/ml, Merck) and Brefeldin A (10 μg/ ml, Merck). Before intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm (BD). Samples were analyzed with a three-laser Cytek Aurora full-spectrum flow cytometer (Cytek Biosciences) and the FlowJo software (Version 10.8, TreeStar, Ashland, OR USA). To minimize spectral spread/background and avoid overly high MFIs, antibodies were used at 0.5 μL per 5×10⁵ cells in 50 μL (1:100). (Higher volumes can push channels toward saturation on full-spectrum systems, using lower volumes preserved clear positive/negative separation while reducing spillover.) Gating was applied sequentially: lymphocytes were identified by FSC/SSC parameters, singlets were selected using FSC-A vs FSC-H, and viable cells were retained by gating on Zombie Aqua− events. All downstream marker analysis was performed on this gated live lymphocyte population:

To reduce figure load while preserving interpretability, plots were limited to the essential bivariate views plus fluorescence-minus-one controls (FMOs, the experimental cells stained with all the fluorophores minus one fluorophore):

• CD3, CD4, CD8, TCRγδ,6B11: CD3 vs target plots, with single-stains and FMOs used to set thresholds. For display, CD3 is shown in the non-stimulated condition; CD8 is shown in the stimulated condition (stimulus-independent marker, representative plot).
• IFN-γ, PD-L1: IFN-γ vs PD-L1 bivariate plots in stimulated samples, with FMOs for each.
• CD11c vs CD69: direct bivariate view (CD11c vs CD69) plus FMOs for both reagents.

TCRγδ (FITC): In CD3 vs TCRγδ, a discrete CD3⁺TCRγδ⁺ population is resolved with clear separation from negatives. FMO-TCRγδ shows no residual cloud in the positive gate, confirming the threshold.

CD3 (eFluor 450): In CD3 vs CD4 and CD3 vs CD8 bivariates, the CD3⁺ compartment is robust with tight CD3⁺ negatives. FMO-CD3 is clean with no events above the positivity threshold.

CD4 (PE-Cy7): In CD3 vs CD4, CD3⁺CD4⁺ cells segregate clearly from CD3⁺CD4⁺. FMO-CD4 shows minimal background and supports a conservative cut-off.

CD8 (Alexa Fluor 700): In CD3 vs CD8 a distinct CD3⁺CD8⁺ subset is cleanly separated from negatives. FMO-CD8 is negative with no clouding in the CD8⁺ region.

PD-L1 (PE): In IFN-γ vs PD-L1 after stimulation, PD-L1 shows a clear up-shift from baseline with a well-defined PD-L1⁺ cluster. FMO-PD-L1 displays minimal background, validating the positivity gate.

IFN-γ (FITC, intracellular): In IFN-γ vs PD-L1, reveals a discrete IFN-γ⁺ population clearly separated from the baseline. FMO-IFN-γ is clean, confirming the threshold.

CD69 (PE-Cy7) In CD11c vs CD69, stimulation yields CD69⁺ shift with a less-defined positive cluster, overall population are resolved with clear separation from negatives. FMO-CD69 is clean and supports a conservative threshold. Note that the apparent CD69⁺ events seen in FMO-CD11c arise from FITC spread/unmixing and are not due to the CD69 reagent itself.

iNKT (6B11, PE): In CD3 vs 6B11 a PE⁺ cloud appears in the expected iNKT region. However, FMO-6B11 shows a similar cloud in Q2, indicating non-specific/background PE signal.

CD11c (FITC): In CD11c vs CD69 the bivariate appears acceptable, but FMO-CD11c shows a cloud within the CD69⁺ gate (Q3) even without CD69 staining. This pattern is not present in the CD69 single-stain, consistent with related spread or imperfect unmixing projecting into the CD69 channel.

Facciotti Group

General and Clinical Pathology
Mucosal Immunology lab

Department of Biotechnology and Biosciences
University of Milano-Bicocca