Facs arial 2 flow cytometer

Single cells isolated from SMGs or submandibular draining lymph node (smLN) cells were incubated with anti-CD16/32 antibody to block non-specific Fc receptor binding, and stained with a panel of fluorescence-conjugated antibodies against CD4, CD8, CD19, CD44, CD45 and CD62 at 4 °C for 30 min. For intracellular and nuclear staining, cells were then incubated with fluorescence-labeled anti-IFN-γ and anti-IL-17A antibodies following fixation and permeabilization. After being rinsed with cold PBS, the stained cells were analyzed with the FACS Arial II flow cytometer (BD, Franklin Lakes, NJ, USA) and FlowJo V10 software. All the antibodies utilized for flow cytometric staining were purchased from BioLegend (San Diego, CA, USA).

The surface markers of P₃ hADSCs were characterized by flow cytometry (BD FACS Aria, Germany) with specific fluorescein isothiocyanate (FITC), phycoerythrin (PE), Peridinin-chlorophyll-protein complex (PerCP), or allophycocyanin- (APC-) conjugated monoclonal antibodies, including CD90, CD105, CD73, CD44, CD29, CD166, CD106, CD45, CD34, CD31, SSEA1, and SSEA4 (BD Biosciences, USA). To evaluate the endothelial phenotype of ADSC cultured on PGA/PLA, the 3D complexes after 7, 14, and 21 days of induction were harvested and digested with 0.25% trypsin for 5 min followed by 0.1% type I collagenase (Sigma, USA) for 15 min to release the cells from polymer, and then the 2D/3D induced cells were tested by mouse anti-human CD34-APC (eBioscience, USA) and mouse anti-human CD31-PE (eBioscience, USA). The protocols were as follows: cells were resuspended with 100 μL buffer (PBS containing 0.1% bovine serum albumin) and incubated for 30 min on ice with the above antibodies. After washing with PBS, the cells were resuspended in the acquisition PBS containing 1% formaldehyde until analysis. In each run, at least 10,000 events were acquired with the FACS Arial II flow cytometer (BD, USA) and the results were analyzed by Flowjo software. HUVECs (purchased from Sciencell, USA) served as the positive control.

To measure the absolute counts of lymphocyte subsets, 50 μl of the sampled blood was transferred to Becton, Dickinson and Company (BD) Trucount tubes (BD, USA; product code: 340334) containing an aliquot of 20 μ six‐colour T, B and NK lymphocytes (TBNK) reagent, including the following fluorochrome‐labelled monoclonal antibodies: anti‐CD45 (PerCP‐Cy5.5), anti‐CD3 (FITC), anti‐CD4 (PE‐Cy7), anti‐CD8 (APC‐Cy7), anti‐CD16⁺CD56 (PE) and anti‐CD19 (APC) (BD, USA; product code: 337166).
The samples were homogenized and incubated in the dark for 15 min at room temperature. Next, the red blood cells in each sample were lysed by adding 450 μl of fluorescence activated cell sorting (FACS) lysis solution (Becton Dickinson, product code: 349202) diluted with ddH₂O at a ratio of 1:10 and incubated in the dark for 15 min at room temperature. The cells were then analysed using a FACS Arial II flow cytometer (BD, San Jose, CA, USA). The definition of the lymphocyte gate for each of the six subsets is shown in Figure 1.
At least 5000 lymphocytes were obtained, and the software Diva was used for data analysis. The absolute counts for each subset were calculated as follows: cell/L = (total number of cells acquired × total number of beads)/(total number of beads acquired × sample volume).

PB mononuclear cells (PBMCs) and SF mononuclear cells (SFMCs) were isolated by density gradient centrifugation. To detect the proportion of CD19⁺CD24^hiCD27⁺ B cells and the expression of RANKL in CD19⁺CD24^hiCD27⁺ B cells of PBMCs or SFMCs, the cells were isolated by density gradient centrifugation and then were stained with mouse monoclonal antibodies as follow: CD19-APC/Cy7 (BioLegend, San Diego, CA, USA), CD24-FITC (eBioscience, San Diego, CA, USA), CD27-APC (eBioscience), RANKL-PE (BioLegend). FMO controls were included. Data was acquired on a FACS Arial II flow cytometer (Becton Dickinson, NJ, USA) and analysed using FlowJo software.
To isolate CD19⁺CD24^hiCD27⁺ B cells, PBMCs were stained with mouse anti-CD19-APC/Cy7, CD24-PE (eBioscience), CD27-APC, then the aimed cell populations were sorted by flow cytometry. Sorted CD19⁺CD24^hiCD27⁺ B cells had a purity of > 95%. These sorted cells were subsequently subjected to reverse transcription-polymerase chain reaction (RT-PCR) or osteoclast differentiation assay.

EdU labeling assay was performed as previously described (43 (link)). In brief, six oysters were cultured with sterile seawater with EdU (Life technologies, 2 mg L^-1) in an aerated tank for 48 h. Cross sections of gill were made as described above. The slides were then fixed with 4% PFA at room temperature for 15 min. After three times washing with 3% BSA in TBS, 0.1% Triton^® X-100 in TBS was used to treat the samples at room temperature for 10 min. CgDM9CP-4 positive cells were stained as mentioned above and after the final three times washing with TBS (containing 3% BSA), 0.1% Triton^® X-100 in TBS was used to treat the samples at room temperature for 10 min. Samples were then incubated with the 1 × Click-iT™ Reaction Buffer (provided with the kit) for 30 min, and washed thoroughly with PBS (3 × 15 min). EdU were analyzed on an FACS Arial II flow cytometer (Becton, Dickinson and Company, USA). For the new generated hemocytes in gill, slices were incubated with DAPI (diluted 1:1,000 in PBST) for 5 min, washed extensively with PBS, mounted with 80% glycerin, and monitored under a Laser Scan Confocal Microscope (ZEISS).