Materials
Human erythroleukemia cell line K562 (China Center for Type Culture
Collection, Wuhan, China) were used as target cells to evaluate the
cytotoxicity of pNK cells. K562 cells were cultured in RPMI 1640 medium
(Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA).
3,3′-Dioctadecyloxacarbocyanine perchlorate (DIO) (1911717; Invitrogen,
San Diego, CA, USA) was used as a lipophilic tracer, and it was
dissolved in dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO, USA) at a
final concentration of 3 mM. Propidium Iodide (PI) (MKCB0899V; Sigma)
was used to detect necrotic cells.
The phenotype and expressions of receptors and cytotoxic granules in NK
cells and subpopulations were analyzed by flow cytometry and it is
listed below. The following antibodies were used: fluorophore-conjugated
monoclonal mouse anti-human antibodies such as PE-cy7-conjugated
anti-CD56 (557747; BD Pharmingen, San Jose, CA, USA), PerCP-conjugated
anti-CD3 (347344; BD Pharmingen, San Jose, CA, USA),
FITC-conjugated
anti-NKG2D
(11e5878;
eBioscience, ThermoFisher, Waltham, MA, USA), PE-conjugated anti-NKp30
(12e3379; eBioscience, ThermoFisher, Waltham, MA, USA), APC-conjugated
anti-NKp46 (17e3359; BD Pharmingen, San Jose, CA, USA), FITC-conjugated
anti-CD158a (556062; BD Pharmingen, San Jose, CA, USA), PE-conjugated
anti-CD158b (559785; BD Pharmingen, San Jose, CA, USA), PE-conjugated
anti-granzyme B (561142; BD Pharmingen, San Jose, CA, USA), Alexa Fluor
647-conjugated anti-perforin (563576; BD Pharmingen, San Jose, CA, USA),
and Alexa Fluor 488-conjugated anti-granulysin (558254; BDPharmingen,
San Jose, CA, USA). All antibodies have been pre-diluted in aqueous
buffered solution containing BSA and ≤0.09% sodium azide.