Brilliant violet 421 annexin 5

Decidual tissues were cut into approximately 0.2-mm³ pieces and then enzymatically digested in 1 mg/ml collagenase V solution at 37 ℃ for 60–70 min. The supernatant was passed through orderly 100-µm and 40-µm cell sieve, and then resuspended in red blood lysis buffer for 10 min. After washing and centrifugation, the leukomonocytes were isolated and incubated in a T75 flask with RPMI 1640 medium for 2 h at 37℃. The non-adherent cells were collected for fluorescence-activated cell sorting with a BD FACSAria II Flow Cytometer (488/633/405), which were incubated with the 7-AAD staining solution (1:400, BioLegend, USA), Brilliant Violet 421 Annexin V (1:200 BioLegend, USA), Alexa Fluor® 700-conjugated anti-human CD45 (1:200 BioLegend, US), APC/Cy7-conjugated anti-human CD56 (1:200 BioLegend, USA), PE-conjugated anti-human CD16 (1:200 BioLegend, USA) and FITC-conjugated anti-human CD9 (1:200 BioLegend, USA) for FACS. Primary dNKs with > 95% purity was obtained for further experiments.
After culture in dNKs medium which contained X-Vivo supplementation (Lonza, USA) of 5% human AB serum and 40 ng/ml IL-15 overnight, the primary dNK cells were divided into two parts and cryopreserved for the further co-culturing or flow cytometric analysis.

Expanded whole BM or c-Kit⁺ BM cells at day 7 and day 14 were stained (1 × 10⁶ cells per sample) with 5 μl of Brilliant Violet 421 Annexin V (Biolegend) and 10 μl of 0.5 mg/ml of propidium iodide (Biolegend)^{27 (link)}. Staining patterns were collected using a FACSAriaIII or FACSVerse (BD) and analyzed with FlowJo (v10.5.3) Software (FlowJo, LLC).