Cytofix cytoperm fixation permeabilization solution kit with golgiplug

Thermo Fisher Scientific supplied mouse anti-E-cadherin, mouse anti-vimentin, mouse anti-catenin, mouse anti-galectin, mouse anti-PD-L1, mouse anti-TGF, mouse anti-NF-κB antibodies, mouse anti-STAT3, and Hoechst (Waltham, MA, USA). eBio-science provided mouse anti-CD163-PerCP, mouse anti-CD206-PerCP, mouse anti-Ki-67-APC, mouse anti-CD68-FITC, and mouse anti-IL-10-FITC (San Diego, CA, USA). BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug™(No. BDB555028, San Diego, CA, USA). PeproTech delivered recombinant mouse IL-4, IFN-, and anti-IL-10 receptor (abIL-10R) inhibitors (Rocky Hill, NJ, USA).
mouse anti-STAT3 (Invitrogen, cat number MA1-13042), rabbit anti-NF-κB (Life technologies, cat number 510500), mouse anti-PD-L1/CD274 (Proteintech, cat number 66248-1-Ig), and mouse anti-TGF (Invitrogen, cat number MA5-15065) were used for immunoblotting, and so were secondary antibodies goat anti-mouse and goat anti-rabbit Alexa® Fluor 555 (Thermo Fisher Scientific, Waltham). For immunoblotting, rabbit anti-actin (LI-COR Biosciences, Lincoln, Nebraska, USA) was used. The anti-mouse PD-L1 checkpoint blocker was purchased from InVivoPlus (B7-H1, Bio cell, USA).

Co-cultured cells were stained with CD3-FITC (clone SP34-2), CD4-APC-CY7 (clone RPA-T4), and CD8-PerceP-CY5.5 (clone RPA-T8). PBMCs from TB patients were stained with CD3-APC-CY7 (clone HIT3a), CD4-APC (clone RPA-T4), CD8-ALEXA700 (clone 53-6.7), CD56-Brilliant Violet 421 (clone HCD56), CD38-PE-CY5 (clone HIT2), and CD14-FITC (clone 61D3). All cells were then fixed with 4% PFA (BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug™). For intracellular staining, fixed cells were permeabilized using a cytofix/cytoperm kit (BD Biosciences) and stained intracellularly with anti-TNF, IFN-γ, and IL-10 antibodies. Cells were acquired (100,000 events) on a BD LSRFortessa® device. The frequencies of stained cells and MFI were estimated using FlowJo 7.10.1 software (Tree Star, Inc, Ashland, OR.).

After incubation and treatment with 1 µM RA, 5μg/ml CHO, 2.5 μg/ml NP3 (RA)-CHO, 2.5 μg/ml NP4-RA, 2.5 μg/ml NP8-CHO, 1.2 μg/ml abIL-10R, and 2.5 μg/ml NP3 (RA)-CHO+1.2 μg/ml abIL-10R for 48 hours, RAW 264.7 cells (1.5 × 104 cells/well in a 12-well plate) were collected with a cell scraper and blocked with 0.5 % BSA in PBS for 45 minutes. To evaluate whether the cells show characteristics of M1 or M2 macrophages, RAW 264.7 cells were labelled with anti-mouse CD68-FITC (1:1000) or CD163-PerCP antibody (1:1000), respectively [11] (link). Next, staining of intracellular IL-10 was performed. For this purpose, cells were fixed in 2% paraformaldehyde (PFA, BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug™) for 15 minutes at room temperature. Next, cells were washed and permeabilized with 0.1% Triton X-100 for 5 minutes and, subsequently, blocked by 0.5% BSA in PBS for 45 minutes. For intracellular staining, permeabilized cells were incubated using a cytofix/cytoperm kit (BD Biosciences) for 1 h. The anti-mouse IL-10-FITC antibody (1:1000) was applied for 60 minutes at 4°C for intracellular IL-10 staining. After a final washing step, the cells were analysed by flow cytometry [11] (link).