Comparative Analysis of NK Cell Activation Markers

Take the flow cytometry FCS files from stimulated and non-stimulated human NK cells. Read these files, identify the NK-cell markers and activation/function markers, and compare stimulated vs non-stimulated samples. Produce a short gating strategy based on the available channels, a summary table comparing marker expression across conditions, and a bar chart showing the clearest stimulation-associated differences.

NK stimulated vs non-stimulated: gating strategy and marker analysis

Three donors (NCI-019, NCI-020, NCI-021) were analyzed under matched stimulated and non-stimulated conditions, for a total of six FCS files. Each file contains 11 fluorescence channels plus scatter and a live/dead stain. Channels were unmixed/compensated upstream (PnTYPE = Unmixed_Fluorescence). All downstream analysis here used an asinh transform with cofactor 150.

Panel and marker roles

Gating strategy

A minimal, panel-appropriate strategy that does not require external compensation matrices:

1. Time / acquisition stability — exclude any acquisition artifacts on Time.
2. Cells — FSC-A vs SSC-A to remove debris.
3. Singlets — FSC-H vs FSC-A (and optionally SSC-H vs SSC-A).
4. Live cells — LIVE/DEAD Blue low (LD⁻).
5. Leukocytes — CD45⁺.
6. Dump (lineage exclusion) — exclude CD14⁺ (monocytes) and CD11c⁺ (myeloid/DC) on a combined dump axis.
7. Non-T — exclude CD3⁺ (removes T and NKT cells).
8. NK cells — CD56⁺ and/or CD16⁺ within the CD3⁻ non-myeloid live CD45⁺ gate. Optional sub-gating: CD56^bright CD16⁻/low vs CD56^dim CD16⁺ to separate cytokine-producing and cytotoxic NK subsets.
9. Activation/function readouts on the NK gate — NKG2D, NKp46, Perforin, Granzyme B (and re-examine CD16 for activation-induced shedding).

With the thresholds used here (asinh cofactor 150; LD < 4.5, CD45 > 4.5, CD14 < 3, CD11c < 3.5, CD3 < 3.5, CD56 > 3 OR CD16 > 4) the NK gate captures roughly 27–73% of CD45⁺ events per sample. Donor NCI-021 (non-stimulated) shows an unusually low CD45⁺ fraction (~14% of live), which is worth checking at the acquisition / sample-prep level before drawing donor-level conclusions.

Marker comparison: stimulated vs non-stimulated

Values are mean ± SD across the three donors. "% positive" uses fixed asinh-space thresholds applied within the NK gate; "MFI shift" refers to the change in median asinh signal.

Granzyme B, NKG2D, Perforin, NKp46, CD56, CD16 are monitored with their respective positive percentages shown in the results. The data show a clear and internally consistent NK activation signature in the stimulated condition.

Interpretation

The data show a clear and internally consistent NK activation signature in the stimulated condition:

• Granzyme B rises from ~4% to ~58% positive.
• NKG2D rises from <1% to ~48% positive.
• Perforin % positive moves modestly (68% → 79%).
• NKp46 and CD56 both shift upward in % positive.
• CD16 drops slightly (90% → 87%).
• CD45 stays at 100% positive, but median signal increases modestly.