Macsmix tube rotator

Immature dendritic cells (DCs) were generated from healthy donor PBMCs by overnight incubation with GM-CSF and IL-4. Mature DCs were generated by overnight incubation of immature DCs with TNF-α, IL-1ß, IL-6, and prostaglandin E-2. Mature DCs were loaded with 1 µg/mL of pooled HLA-restricted peptides and incubated for 4 h at 37 °C in a MACSmix™ Tube Rotator (Miltenyi Biotec). CD8⁺ T cells were isolated from autologous PBMCs using the EasySep™ Human CD8⁺ T Cell Enrichment Kit (STEMCELL Technologies). Following incubation, peptide-loaded DCs were irradiated at 4000 RAD. Lines were generated (10/donor) by combining irradiated DCs with CD8⁺ T cells and IL-21. Lines were incubated at 37 °C and maintained every 2-3 days with CTL, IL-2, IL-7, and IL-15. 10-14 days following line generation, stimulation 2 was carried out by combining irradiated PBMCs from the same donors with cells from stimulation 1, pooled peptides at 1ug/mL, and IL-21. This process was repeated for a total of three stimulations. In the REP, CD8 + T cells were expanded in the presence of anti-CD3 Abs (100 ng/mL), anti-CD28 Abs (100 ng/mL), IL2 (20 IU/mL), IL7 (10 ng/mL), IL15 (10 ng/mL), irradiated mixed PBMCs from the three healthy donors and TM-LCL lines. The cell culture medium was half-replenished every 3 days up to 14 days of culture.

The Nkx2-2enh::CD14 transgene-expressing cell fraction was enriched by MACS^{29 (link)} from in vitro differentiation cultures. Developing embryoid bodies were dissociated with Accumax at 37 °C at 400 rpm shaking for 10 min. Cells were labelled with anti-CD14-APC primary at 1:50 concentration (Supplementary Table S3), and after a wash, mouse anti-APC MicroBeads (130-090-855, Miltenyi) at 1:5 concentration in MACS buffer for 15 min each at 4 °C in a Miltenyi Biotec MACSmix Tube Rotator. Pre-sort, flow through and eluate fractions were analysed using a BD Fluorescence-activated cell sorting (FACS) Canto II machine (NIHR Guy’s and St. Thomas’ Biomedical Research Centre). DAPI was added at 1 µg/ml to exclude the dead cells just before the analysis. 10,000 cells were analysed using the FlowJo software (BD Biosciences). Live single cells were gated based on forward and side scatter (FSC and SSC) parameters, as well as DAPI absorbance. Unstained cells were used to gate for an APC-positive fraction.

Colonic cell isolation was performed as previously published with a few modifications (20 (link)). Briefly, colons were removed, cleaned of mesentery and feces, and opened up longitudinally. They were then transversely cut into small pieces of ~1 cm in length, and intraepithelial and LP fractions were extracted following the manufacturer’s protocol (LP dissociation kit, a MACSmix tube rotator, Miltenyi Biotec). Following two rounds of predigestion solution using the MACSmix tube rotator, the IEL fraction was generated. The remaining tissue was washed to remove the EDTA, and supernatant was again pooled with the IEL fraction (third wash). The colon pieces were then incubated in digestion solution (containing enzymes) at 37°C and gently shaken as mentioned using the MACSmix tube rotator. Finally, tissue pieces were disrupted using the m_intestine_01 program of the gentleMACS dissociator and passed first through a 100-μm filter and then through a 70-μm filter to obtain the LP fraction.
For the small intestinal IEL fraction, a similar method was followed. For mesenteric lymph node (MLN), lymph nodes were carefully isolated and made free of extra fat and then physically mashed gently using the plunger of a 5-ml syringe while passing through a 70-μm filter to obtain single-cell suspensions.