Anti ki 67 pe

EDTA anticoagulated peripheral blood was used for phenotype analysis by flow cytometry. In detail, 200 μL of whole blood was lysed in 2 mL of VersaLyse at room temperature for 15 min. Then, cells were washed twice with FACS buffer, followed by resuspension in the appropriate antibody preparation and incubated for 20 min at 4°C. For intracellular staining of Ki-67 expression, we used permeabilization and fixation method using eBioscience perm/fix kit (ebioscience cat no. 88-8824-00). The following monoclonal antihuman antibodies were used in appropriate concentrations to stain cells: anti-CD45-Krome orange (Beckman Coulter, cat no. 96416), anti-CD14-PC5.5 (Beckman Coulter, cat no. A70204), anti-CD 19-APC-Alexa fluor 750 (Beckman Coulter, cat no.A94681), anti-CD27-PE (BD Pharmingen, cat no.555441), anti-CXCR3-APC (BD Pharmingen, cat no. 561732), anti-CXCR4-APC (BD Pharmingen, cat no. 560936), anti-CD95-APC (BD Pharmingen, cat no. 558814), anti-ki-67-PE (Ebioscience Cat no. 12-5699-42), and anti- IgD-FITC (BD Pharmingen, cat no. 555778). After staining, the cells were analyzed by 10-color flow cytometry (Navios, Beckman Coulter), with at least 20,000 CD19+ events collected for each analysis.

Cell suspensions were prepared from the spleen or a mixture of all of the placentas and uteri, except one placenta for cytokine assays and one for histopathology assays. Mononuclear cells of the spleen or the mixture of placentas and uteri were obtained using mouse lymphocyte separation liquid (TBD Tianjin hao yang, China) with the density of 1.0830 ± 0.0001 g/ml (20°C) and washed in cold PBS. Cells were incubated with anti-CD4-Percp-cy5.5, anti-PD-1-PE, or anti-CTLA-4-PE at 4°C in the dark for 30 min, and then were washed twice. All of the above mAbs were from BD Pharmingen (United States). For intracellular Foxp3, pSmad3, and Ki-67 staining, cells were fixed and permeabilized in Fix/Perm buffer (eBioscience, United States) for 30 min and subsequently incubated with anti-Foxp3-APC, anti-pSmad3-PE, or anti-Ki-67-PE, in accordance with the manufacturer’s instructions. These mAbs were from eBioscience (United States). After washing three times with PBS, the cells were analyzed with a BD FACSCantoTM II flow cytometer (Becton Dickinson, United States) using BD FACSDiva 7.0 software.

MIN6 cells were pretreated, washed thrice with PBS, collected, and stained using an EdU assay kit (Beyotime Biotechnology) or anti‐Ki67‐PE (eBioscience) antibody. Data were obtained using CytoFLEXS flow cytometer (BD Biosciences) and analyzed using CytExpert.

Cells were collected and subjected to three washes with PBS to ensure cleanliness and integrity. For the cell surface marker CD8, flow cytometry antibodies (PerCP, eBioscience) were introduced and incubated away from light at room temperature for 30 mins. Subsequently, the cells underwent a thorough three-time wash. For intracellular marker staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then stained with fluorochrome-conjugated antibodies, including anti-ki-67-PE, anti-IFN-γ-PE, and anti-TNF-α-FITC, all sourced from eBioscience, for 30 mins. Post-staining, 100 μl of PBS was added to each tube for cell resuspension and detected by BD FACSCalibur flow cytometer. The acquired data were quantified using FlowJo software (v10).