Ficoll paque plus

EDTA-coated tubes, containing human blood samples, were centrifuged at 800g for 15 min at 24°C with brakes off. The plasma layer on top was removed. An equal amount of serum-free cell culture medium (TexMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) was added and thoroughly mixed. Seven parts of the diluted sample was carefully layered on top of three parts of Ficoll-Paque Plus (MilliporeSigma, Burlington, USA). Tubes were centrifuged at 800g for 30 min at 24°C with brakes off. The layer of PBMCs at the interface was transferred into a fresh tube. Cells were washed twice with 1X phosphate buffered saline (PBS; w/o Ca/Mg, Gibco, Billings, USA) by centrifuging at 800g for 10 min at 24°C. Cells were counted with a hemocytometer and viability was determined with Trypan Blue. PBMCs were resuspended in CryoStor^® CS10 (STEMCELL, Vancouver, Canada) and cryopreserved in liquid nitrogen.

Human samples (gingival biopsies and saliva) were obtained with informed consents from healthy and HIV+ (PLWH) cohorts under a protocol approved by the University Hospitals Cleveland Medical Center Institutional Review Board, complying with all relevant ethical regulations^{4 (link)}. Participants of both sexes were enrolled. The characteristics of enrolled participants for obtaining gingival biopsies and saliva are described in Supplementary Table 1. Gingival biopsies were processed fresh for flow cytometry. Discarded palatine tonsils were obtained from tonsillectomy surgeries performed at University Hospitals Cleveland Medical Center through the Histology Tissue Procurement Facility following a separate IRB-approved protocol (Non-Human research). A single-cell suspension of gingival tissues and tonsils was prepared by Collagenase 1A digestion (0.5 mg/ml; Sigma C9891), with subsequent Ficoll-Paque PLUS (GE17-1440-02; Millipore Sigma) centrifugation at 900 g and washing with PBS. Tonsil cells were processed fresh for HTOC cultures. Some tonsil cells were processed fresh for CD4 cell purification for cell culture or flow cytometry. Saliva samples were collected in sterile tubes and stored at −80 °C until processing for metabolome and ELISA analyses.

The frequency of DNA DSBs within peripheral blood mononuclear cells (PBMC) was determined by microscopic quantification of focal accumulations of γ-H2AX and 53BP1^{19 (link)}, two marker proteins indicative for the presence of a DSB. For this purpose, PBMCs were isolated from blood samples by Ficoll Paque Plus (Merck, Darmstadt, Germany) density centrifugation. PBMCs were washed twice with PBS, fixed in 70% ethanol and stored at − 20 °C. Immunofluorescence staining and DSB foci analysis was performed as described previously^{19 (link),20 (link),42 (link)}. Using a Zeiss Axioimager Z2 epifluorescence microscope of the MetaSystems (Altlussheim, GER) ISIS imaging system equipped with appropriate single and dual-band filter sets co-localizing γ-H2AX + 53BP1 foci were counted in 100 well separated and morphologically intact cells by an experienced investigator (HS).

Cryopreserved PBMC from 23 patients with UC part of the Mater Hospital IBD biobank, Brisbane, Australia, were assessed by flow cytometric analysis (Table 1). Age‐ and gender‐matched PBMC from healthy donors were included as controls. Blood samples were collected as part of the Mater IBD biobank (Mater HREC approval AM/MML/24730). PBMC from patients receiving anti‐TNF therapy were excluded from our analysis because of direct effect of TNF‐α on Treg.⁴⁵ (link) Before staining, cryopreserved samples were thawed and incubated in RPMI 1640 containing 10 µg mL⁻¹ DNAse I (Roche, Basel, Switzerland) at 37°C for 1 h to prevent cell clumping and debris. PBMC from healthy controls were freshly isolated from volunteers at QIMR Berghofer for transcriptome, proteomic and functional studies. Ethics approval was obtained from the human research ethics committee QIMR Berghofer, Brisbane, Queensland, Australia (HREC #P2058). In all cases, PBMC were isolated using a Ficoll‐Paque Plus (Merck, Kenilworth, New Jersey, USA) density gradient centrifugation from blood and a written informed consent was obtained from volunteers.

Blood samples were collected in EDTA containing tubes from clinically healthy and regularly slaughtered cattle (n = 3), selected in a CBPP free area (Italy). PMNs were isolated by density gradient using Ficoll Paque Plus (Merck KGaA, Darmstadt, Germany), according to manufacturer's instructions. Cell precipitate was treated with a hypotonic lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.12 mM EDTA) to remove red blood cells. Then, PMNs were re-suspended in RPMI media (Merck KGaA, Darmstadt) to a concentration of 10⁶per ml with a viability ≥ 90%, which was determined using an automated cell analyser (Vi-Cell, Beckman Coulter).
Re-suspended PMNs were seeded at a density of 5 × 10⁵ cells per well (500 μl) in 24-well flat-bottom plates (Falcon, Corning incorporated) with or without Mmm (5 × 10⁷cells per well) (500 μl) to obtain a multiplicity of infection (MOI) of 100. The plates were incubated at 37°C in 5% CO₂ in mild shaking and cell suspensions were sampled at 30 min and 1, 2, 3, 6, and 18 h after Mmm exposure. Each sample (exposed PMNs and not exposed PMNs) was assessed in duplicate for every time point considered.