Facs symphony a3

Isolated cells were incubated with Ghost Dye Red 780 Viability Dye (TONBO biosciences) for 30 minutes on ice, in the dark, then washed and stained with FcBlock and surface antibodies for 45 minutes to 12 hours at 4°C in the dark. Antibodies used are documented in Table S1. For intracellular staining of cytokines, cells were treated with GolgiStop (containing monensin) for 4 hours and fixed for 20 minutes using fixation/permeabilization solution (BD, 554715) prior to intracellular staining for 45 minutes. Analysis was performed on a BD Fortessa X-30 and BD FACSSymphony A3 cytometers and using FlowJo v10; gating schemes are shown in relevant supplementary figures. Antibodies used can be found in Table S1. For fluorescence associated cell sorting: 15,000 live CD45⁺ CD19⁺ CD11b⁺ and live CD45⁺ CD19⁺ CD11b⁻ were sorted on a BD FACS Aria II into RPMI with 40% FBS. Cells were pelleted and resuspended in RNA lysis buffer (Invitrogen; 46-6001) for RNA isolation. Adipose B cells were pooled from 2 mice per sample to account for biological variability.

WST-1(4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene-disulfonate (ROCHE/CELLPRO-RO) colorimetric assay was carried out to determine the effects of NIR laser irradiation (808 nm) on cell proliferation at the above indicated condition. The assay was performed by 48-well plates, with seeding of 4T1 (5 × 10³ cells/well), A549 (1 × 10⁴ cells/well), RAW264.7 (3 × 10⁴ cells/well) cells on PLGA, PLGA-GO1% and PLGA-GO2% scaffolds. After each NIR application, cells were cultured for additional 24 h s and 30 μL per well of WST-1 solution were added to the culture medium and incubated for 2 h at 37 °C and 5% CO2. Absorbance was subsequently determined using Cytation 3 Cell Imaging Multi-Mode Reader (Biotek) applying the wavelengths 450 nm for measurements and 650 nm for reference. All experiments were conducted in two wells for each condition and replicated at least three times. Cell proliferation was calculated by comparing the absorbance values of the samples after background subtraction. Cell viability was expressed as the percentage of cells seeded on PLGA. Cell viability of human PBMCs was assessed by staining PBMC harvested from the different culture conditions and by staining cells with E-Fluor 780 (Thermofisher) to exclude dead cells. Stained cells were then acquired at FACS Symphony A3 within 1 h and results analyzed using Flowjo 10.7v (BD Biosciences).