Cd49b pe

Resting NK cells (2 × 10⁵ cells/mL) and mDCs (1 × 10⁶ cells/mL) were co-cultured at a ratio of 1:5 in the 96-well U-bottomed plate for 2 d. The cultured cells were then harvested, and the supernatant was stored for cytokine ELISA. Then, the cells were resuspended in FACS buffer, which was stained with different surface markers, including Live/Dead-Amcyan (Thermo Fisher Scientific), CD49b-PE (BD Pharmingen^TM), CD69-AF700 (activation marker for NK cells; BD Pharmingen^TM), CD11c-PE/Cy7 (Thermo Fisher Scientific), CD40-BV605 (Thermo Fisher Scientific), and intracellular cytokine marker IFN-γ-APC/Cy7 (BD Pharmingen^TM). The cell phenotypes and the frequency of IFN-γ were determined using the Becton-Dickenson LSR flow cytometer and analyzed using the Flowjo Software Program (Tree Star Inc., OR, USA).

Immature DCs (iDCs, 2 × 10⁵ cells/mL) and ANK cells (4 × 10⁵ cells/mL) were co-cultured at a ratio of 1:2 in the 96-well U-bottomed plate. After 2 d of incubation, the cultured cells were harvested. The supernatant was stored for cytokine ELISA and the cells were resuspended in FACS buffer and stained with different surface markers, including Live/Dead-Amcyan (Thermo Fisher Scientific), CD49b-PE (BD Pharmingen^TM), CD69-AF700 (BD Pharmingen^TM, Franklin Lakes, NJ, USA), CD11c-PE/Cy7 (Monoclonal Antibody N418; Thermo Fisher Scientific, Waltham, MA, USA), CD40-BV605 (clone 3/23; BD Biosciences, Franklin Lakes, NJ, USA), and CD86-FITC (clone GL1; BD Pharmingen^TM). Flow cytometry data were acquired using a Becton-Dickenson LSR flow cytometer and analyzed using the Flowjo Software Program (Tree Star Inc., Ashland, OR, USA).

The primary anti-human or anti-mouse antibodies (CD29-PE, CD49a-PE, CD49b-PE, CD49c-PE, CD49d-FITC, CD49e-PE, CD49f-PE, CD51/61-PE, CD44-APC, CD45-FITC, CD47-PE, CD324-PE, CD325-PE, CD326-PE, CD54-PE, CD106-FITC, CD62E-APC, CD62L-APC, CD15s, isotype controls) and secondary antibodies were all obtained from Becton Dickinson (BD) Pharmingen™. Human interleukin-1 beta (IL-1β) was purchased from Cell Signaling Technology, Inc. 17β-estradiol (E2) and progesterone (P4) were purchased from Shanghai Yuanye biological technology Co., Ltd Mifepristone (RU-486) was purchased from Shanghai New Hualian pharmaceutical Co., Ltd with purity > 98%. Metapristone was synthesized by our laboratory with purity > 98%. Calcein-AM was obtained from Dojindo Molecular Technologies, Inc.

BALF cells were resuspended in FACS buffer (PBS 1% FCS). Lungs were perfused with ice-cold PBS to remove excess blood before single cell suspensions were obtained using collagenase. Briefly, whole lungs were digested with RPMI containing collagenase D (1 mg/mL) and DNase I (Roche, Mannheim, Germany) and cells were washed and recovered by centrifugation. Erythrocytes were lysed by incubation with RBC lysis buffer. To avoid non-specific binding of Abs to FcRγ, FACS Buffer containing anti-mouse CD16/32 mAb (Mouse BD Fc Block) (2.4G2, BD) was added to all primary stains. Cells were labeled with fluorophore-conjugated antibodies at pre-optimized dilutions to CD3-FITC, CD4-PE, CD8-PE, CD49b-PE (NK/NK T marker) and CD69-FITC (all from Becton Dickinson) for 1 h at 4°C and then washed twice in FACS buffer and resuspended in a final volume of 0.5 ml of FACS buffer. Data was acquired on a BD FACSCalibur flow cytometer (Becton Dickinson) and typically up to 10⁵ viable cell events were collected for analysis. A strict gating strategy was used to determine different immune cell populations as follows: single cell gate (FSC-H vs FSC-A), live cells (propidium iodide exclusion), granularity/size cell gate (FSC-A vs SSC-A) and specific surface marker gates. Flowjo software (version 7.2.4, Tree Star, OR) was used to generate plots for data analysis.