Fig 2: Expressions of CD8, IFNg, and FoxP3 in tumors recovered from two cohorts (B3 and B9) comparing aPD1/ADA gel and saline injections (a third cohort had poor RNA quality (RIN <7) and therefore excluded). RT-qPCR analyses were performed for (A) CD8a, (B) IFN?, and (C) IFN? relative to FoxP3 (ddCt IFN?-FoxP3); lower dCt indicates higher expression; Expressions of the same genes from two other cohorts (B5 and B7) comparing aPD1/ADA gel and aPD1 gel-treated tumors resulted in insignificant differences in (D) IFN? and (E) IFN? relative to FoxP3, but significant difference in (F) IL-17. RNA were extracted from tumors processed into single cell suspensions using Miltenyi dissociation kits in a GentleMACS. Expressions of the genes were probed with TaqMan primers and normalized to actin. Purities of the RNA were determined using Agilent Nano RNA chip. Significance was determined using unpaired two-tailed t-test with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Biochemical features of hepatocytes isolated with the gentleMACS Perfusion Technology. (A) Comparative analysis of Albumin (Alb) and hepatocyte nuclear factor 4a (Hnf4a) mRNA expression in hepatocytes isolated either by the gentleMACS Perfusion Technology or conventional isolation procedure. The RNA was subjected to a standard RT-PCR using the following primers: Alb-for: 5'-ggt ctc atc tgt ccg tca gag-3', Alb-rev: 5'-gga aga cat cct tgg cct cag-3', Hnf4a-for: 5'-cca aga ggt cca tgg tgt tt-3', Hnf4a-rev: 5'-ccg agg gac gat gta gtc at-3', Actb-for: 5'-ctc tag act tcg agc agg aga tgg-3', and Actb-rev: 5'-atg cca cag gat tcc ata ccc aag a-3'. Cycle conditions were: 5 min initial denaturation at 95 °C, 1 min at 95 °C, 1 min annealing at 60 °C (Actb, 25 cycles; Hnf4a, 35 cycles) or 64 °C (Alb, 30 cycles), 3 min extension at 72 °C, and final elongation at 72 °C for 10 min. Amplicons were separated in 1.6% (Alb) or 2% (Actb, Hnf4a) 1 × TBE-buffered agarose gels containing ethidium bromide and visualized using gel imager Gel iX20 (Intas Science Imaging Instruments GmbH, Göttingen, Germany). (B) Western blot analysis of protein extracts and supernatants isolated from cultured hepatocytes isolated with the gentleMACS Perfusion Technology that were stimulated with recombinant human IL-1ß (#130-093-895, Miltenyi Biotec) or left untreated (control). (C) Comparative analysis of IL-1ß sensitivity of hepatocytes isolated with the gentleMACS Perfusion Technology or the conventional isolation protocol. Antibodies used were directed against estrogen receptor-a (ER-a) (#MA1-27107, ThermoFisher), Lipocalin 2 (LCN2) (#AF3508, R&D Systems), or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#sc-32233, Santa Cruz Biotech.).

Fig 4: Nasal polyp digestion protocols for mass cytometry. (A) Nasal polyps were minced, and submitted to one of the following digestion protocols: 1. incubation at 37°C in a water bath for 40 min in collagenase solution; 2. incubation on ice for 60 min in protease solution; and 3. incubation for 53 min in enzyme mix (A, D, and R enzymes) in GentleMACS® (Miltenyi). Single cell suspensions obtained were used for labeling according to the Maxpar Direct Immune Profiling Assay (MDIPA) protocol (figure created with BioRender.com). (B) Mean metal intensity (MMI) of CD3, CD45 and CD11b positive live cells obtained with each protocol by mass cytometry. Bars represent mean of three independent experiments. (C) Percentage of CD3, CD45 and CD11b positive live cells obtained with each protocol by mass cytometry. Lines represent mean of three independent experiments.