Goat anti mouse igg microbeads

A mononuclear fraction of white blood cells (WBC) was isolated from the whole heparinized blood from 6 healthy adult pigs using a density gradient technique (Histopaque 1.077, Sigma-Aldrich, St. Louis, MO). Subsequently, a CD14-positive cell subset was selected by indirect magnetic labeling on QuadroMACS™ cell separator (Miltenyi Biotec, Gladbach, Germany) using monoclonal antibody against CD14 (clone MIL2, AbD Serotec, Oxford, UK, 10 μl per 10⁸ cells). CD14-positive cells were captured by goat anti-mouse IgG MicroBeads (Miltenyi Biotec, Gladbach, Germany). The cell subset purity was assessed using flow cytometer LSRFortessaTM (BD Biosciences, San Jose, CA) and was more than 95% in all cases. CD14-positive monocytes, approximately 0.5 × 10⁶ cells per well in 24-well plates, were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Paisley, UK) supplemented with antibiotics (100,000 IU/l penicillin; 10 mg/l streptomycin; 4 mg/l gentamicin) and 10% (v/v) heat-inactivated porcine serum (PAA Laboratories, Pashing, Austria) at 37°C in an atmosphere with 5% (v/v) CO₂. After 6 days of cultivation, monocyte-derived macrophages (MDMF) were prepared [30 (link)].

Single-cell suspensions of lymphocytes were prepared from spleen tissues of uninfected birds using Histopaque-1083 (Sigma-Aldrich) density-gradient centrifugation. CD4⁺ T cells were isolated by magnetic cell sorting using mouse anti-chicken CD4 antibodies and goat anti-mouse IgG microbeads (Miltenyi Biotec). After each antibody treatment, cells were washed three times with PBS containing 0.5 % BSA. At each wash, the cell suspension was centrifuged at 450 g for 10 min. Positively stained cells were sorted through an AutoMACS Pro Separator (Miltenyi Biotec). Purity of the sorted cells was confirmed to be >99 % by flow cytometry after labelling with monoclonal anti-goat/sheep IgG–fluorescein isothiocyanate (Sigma) antibody (data not shown).

Monomorphonuclear cells (MCs) were isolated from the jejunal lamina propria (LP) as described above. APCs were further enriched from the MC fraction by immunomagnetic cell separation (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). LPMCs were labelled with an anti-MHCII mAb (clone MSA3, IgG2a) and goat anti-mouse IgG microbeads (Miltenyi Biotec). MHCII⁺ cells were retained within a LS column (Miltenyi Biotec) placed in a magnetic field. After washing, the cells were flushed out and stained with anti-SIRPα-DyLight649 (clone 74-12-15, IgG1; DyLight649 conjugation kit, ThermoScientific) and anti-CD16-FITC (IgG1; AbD Serotec, UK). SytoxBlue (1 mM; Invitrogen) was used to stain dead cells according to the manufacturer’s instructions. MHCII⁺SIRPα⁺CD16^hi (CD16^hi) and MHCII⁺SIRPα⁺CD16⁺ (CD16⁺) LPMCs were FACS purified (FACS AriaIII; BD Biosciences, Erembodegem, Belgium). Post-sort analysis revealed a >95% purity of both populations. Sorted cells were stained with anti-human CD68 mAb (IgG2b; eBioscience, Y1/82A) and anti-mouse IgG_2b-AF594 (Invitrogen, A21145). Nuclei were counterstained with Hoechst (10 µg/ml). Cells were imaged with a fluorescent microscope.

1 × 10⁷ cells from 4T1 spheres, as well as monolayer culture and MDAMB-231 spheres, were incubated with primary antibody against CD24 according to the manufacturer's procedure mentioned in CD24 Microbead Kit (MiltenyiBiotec). Following that, goat anti-mouse IgG microBeads (Miltenyi Biotec) were added to the labeled cells and MiniMACS columns (Miltenyi Biotec) were used to magnetically separate the cells. CD44 microbeads (Miltenyi Biotec) were added to acquire CD24⁻ cells at 4 °C for 15 min. Cells were again washed and magnetically separated. The CD44⁺/CD24^−/low CSC population was collected for further experiments.

Following PBMC isolation as described above, depletion of CD4, CD8, or γδ T cells was performed to allow better characterization of the Tp9-specific cellular immune response. Monoclonal antibodies to bovine CD4, CD8, TCR δ chain (Supplementary Table 2) were used with MACS® MicroBeads (Miltenyi Biotec) for depletion by following the manufacture's protocol. Briefly, cells were incubated with the monoclonal antibody/ies specific for markers of the cell population(s) to be depleted (1 μg mAb/10⁶ cells) for 15 min at 4°C in MACS buffer (PBS without ${\text{Ca}}_2^{+}$ and ${\text{Mg}}_2^{+}$ , pH 7.0, 0.5% BSA, and 2 mM EDTA). Subsequently, cells were washed twice in MACS buffer and incubated with goat anti-mouse IgG MicroBeads (20 μl MicroBeads/10⁷ cells) (Miltenyi Biotec) for 15 min at 4°C. Cells were then washed twice in MACS buffer and passed through a MACS LS column (Miltenyi Biotec). Efficiency of depletion was evaluated by flow cytometry using the Guava® easyCyte™ HT system (Millipore). CD4 depleted cells, CD8 depleted cells, or CD4/γδ depleted cells were used for IFNγ ELISpot as described above.

HepG2 or MHCC-LM3 cells were first labeled with primary antibody for CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany) and then incubated with goat anti-mouse IgG microbeads (Miltenyi Biotec) according to the manufacturer’s protocols. Cells were then sorted magnetically using MACS LS columns (Miltenyi Biotec). After dead cells were excluded from the sort via an electronic gate, cells expressing CD133 were collected through a sort gate. In addition, phycoerythrin (PE)-conjugated CD133 (BD PharMingen, San Jose, CA, USA) were used in the experiment. The processed cells were incubated in phosphate-buffered saline (PBS) containing 2% FBS followed by PE-conjugated antibodies. Isotype-matched mouse immunoglobulins served as controls. The samples were analyzed using a FACSCanto II analyzer flow cytometer (BD Biosciences, San Jose, CA, USA).

Separation of Th17-cells was performed by using a modified protocol by KOL et al. [27 (link)–29 ]. PBMCs were blocked with Human TruStain FcX™ (BioLegend®, California, USA) and subsequently marked with the following antibodies: mouse anti dog CD8 alpha (1:5 diluted with staining buffer), mouse anti dog CD11b (1:11 diluted with staining buffer), mouse anti canine CD21 (first dilution: 1:5 with staining buffer, second dilution: 1:11 with staining buffer) and goat anti-mouse IgG microBeads (1:5 diluted with staining buffer; MACS Miltenyi Biotec, Germany). Afterwards, the cell suspension was separated using the “Deplete” program of autoMACS® Pro Separator (Miltenyi Biotec GmbH, Germany). Thus, undesired cells (CD8 alpha+, CD11b+, CD21+) were sorted out by magnetic columns and the target population of CD3+ and CD4+ cells could be assembled. All antibodies were purchased from Bio-Rad Laboratories, Inc., California, USA.