Alexa fluor 700 conjugated anti human cd45

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.

Initially, JEG-3 (1×10⁵) cells were labeled with Red fluorescence CMPTX dyes and seeded into a 12-well plate. Subsequently, NK92 cells (1×10⁶) were added to each well. After 24 h of culture, co-culture cells were harvested, blocked with human IgG in RPMI1640 with 1% FBS, and stained with Alexa Fluor^® 700-conjugated anti-human CD45 (1,200 BioLegend, US), APC-conjugated anti-human HLA-G (1,200 BioLegend, US), and 7-AAD staining solution (1,400, BioLegend, USA) for 30 min. Subsequently, the data were collected using a Deflex B5-R3-V5 Flow Cytometer (405/488/638) and analyzed using the FlowJo_v10.6.2 software. All the antibodies used for this study are described in Supplementary Table 2.