Spectramax id5 multi mode microplate reader

NAGLU activity was determined using the NAGLU substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-α-D-glucopyranoside (Toronto Research Chemicals, North York, ON, Canada, M333800) and HEXB activity was determined using the HEXB substrate, 4-methylumbelliferyl-N-acetyl-β-glucosaminide (EMD Millipore Chemicals, 474502). For NAGLU activity, reaction mixtures consisted of 2.5 μL of cell lysates, 2.5 μL of 4 mM NAGLU substrate in 200 mM acetate buffer pH 5.6 with 0.01% Triton. For HEXB activity, cell lysate was diluted 1000-fold using PAD buffer (10 mM sodium phosphate, pH 5.8, 0.02% sodium azide, 0.1 mM dithiothreitol, 0.1% Triton X-100), reaction mixtures consisted of 2.5 μL of cell lysates and 2.5 μL of 2.5 mM HEXB substrate in PAD buffer. After incubation for 2 h at 37 °C, the mixtures were quenched by adding 65 mL of 500 mM glycine/sodium carbonate pH 10.6. The reaction mixtures were transferred into a 384 well plate. Fluorescence measurements were obtained using a SpectraMax^® iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) at excitation and emission wavelengths of 360 nm and 450 nm, respectively.

Cells (500 cells/well) were seeded in 96‐well plates and left to adhere overnight. When untreated control wells reached 80% confluency, cells were fixed with 10% formalin and stained with 0.5% crystal violet. The stain was solubilized with 20% MeOH, and absorbance was measured at 570 nm on the SpectraMax iD5 Multi‐Mode Microplate Reader (Molecular Devices: San Jose, CA).

Poly (T)-tagged DNA used in these studies were labeled with FAM at 3′-terminal (Supplementary Table 2). FAM-labeled DNA were first absorbed to AuNPs surface following the protocol as above described. The FAM-DNA-AuNP conjugates were resuspended in 0.01 M phosphate buffer (0.3 M NaCl, pH 7.4) or 10 mM HEPES buffer. The surface density of the DNA-AuNP conjugate was quantitated according to the published protocol^{56 (link)}. First, β-mercaptoethanol (ME, catalog number: 63689) was added (20 mM final concentration) to the FAM-labeled DNA-AuNPs solution and incubated overnight with shaking at room temperature. Then, the mixture was centrifugated at 15 °C at 12,400×g for 20 min and the supernatant was collected. The released FAM-DNA probes were in the supernatant and the fluorescence was measured by SpectraMax iD5 Multimode Microplate Reader (Molecular Devices, CA, USA). The fluorescence intensity was converted to the molar concentration of DNA probes by interpolation from a standard linear calibration curve that was prepared with known concentrations of same FAM-DNA probes at same buffer condition and ME concentration. AuNPs concentration was determined via the absorption of UV-vis spectra and finally the number of DNA on each AuNP could be calculated.

MC38 tumors without IR and three days post-IR were excised from WT and Sirpα^−/− mice. After being weighed, tumors were minced followed by culturing in RPMI-1640 with 10% FBS. After 24 h, the culture supernatants (tumor-conditioned medium) were collected and tested for the presence of MCP-1 and KC chemokines by a multiplex assay (BioLegend), and the capability of driving monocyte/MDSC or PMN chemotaxis by in vitro chemotaxis assays^{49 (link),51 (link)}. Ly6C⁺ monocytes/MDSCs and Ly6G⁺ PMNs (1 × 10⁶ each) isolated from WT mouse bone marrow^{52 (link)} were labeled with CFSE and placed into the upper chamber of the transwell device in a 24-well plate. The collected tumor-conditioned medium (0.5 ml) was added into the lower chamber followed by incubation at 37 °C for 2 h. Chemotaxis of monocytes/MDSC and Ly6G⁺ PMN into the lower chamber was quantified by a SpectraMax iD5 Multi-Mode Microplate Reader with SoftMax Pro 7 Software (Molecular Devices) and calculated against the total cells loaded. Control experiments were performed using medium without culturing tumor tissues.