Gentlemacs dissociator system

Necropsy was performed as previously described (Gideon et al., 2015 (link); Lin et al., 2009 (link), 2013 (link); Maiello et al., 2018 (link)). Briefly, an ¹⁸F-FDG PET-CT scan was performed on every animal 1–3 days prior to necropsy to measure disease progression and identify individual granulomas. At necropsy, monkeys were maximally bled and humanely sacrificed using pentobarbital and phenytoin (Beuthanasia; Schering-Plough, Kenilworth, NJ). Individual granulomas previously identified by PET-CT and those that were not seen on imaging from lung and mediastinal lymph nodes were excised for histological analysis, bacterial burden, and other immunological studies. TB specific gross pathologic lesions and overall gross pathologic disease burden were quantified using a previously published method (Maiello et al., 2018 (link)). The size of each granuloma was measured by pre-necropsy scans and at necropsy. Granulomas were enzymatically dissociated using the gentleMACS dissociator system (Miltenyi Biotec Inc) to obtain a single suspension for enumerating bacterial burden and for single cell RNA-sequencing (scRNA-seq) on the Seq-Well platform.

To achieve consistent mechanical disruption and proteolytic enzyme exposure across a fragment of tissue, large synovial specimens (> 150 mg) were cut into small fragments (~ 2 mm³) using surgical scissors. Mechanical disruption of arthroplasty samples (six tissue fragments totaling ≥ 500 mg) was carried out by a gentleMACS dissociator system (Miltenyi) with the m_Spleen 04.01 setting, which involves churning and shredding in a sterile disposable tube. Enzymatic digestion was performed in RPMI medium at 37 °C for 30 min. A large volume of 5% FBS/RPMI was then added to terminate the enzymatic reaction. The tissue was ground through a 70-μm filter using the flat plastic end of a 3-ml syringe plunger to disperse the remaining intact tissue and dispense dissociated cells.

Freshly collected decidua basalis was washed with cold PBS and processed using methods previously described (37 (link)). Briefly, tissue was minced and dissociated in RPMI containing 1 mg/mL of Collagenase from Clostridium histolyticum (Sigma Aldrich, St. Louis, MO) and 1 μg/mL Deoxyribonuclease I from bovine pancreas (Sigma Aldrich, St. Louis, MO), using the GentleMACS Dissociator system (Miltenyi Biotec Inc., San Diego, CA, USA). Homogenates were then filtered through a 100-μm filter; red blood cells were lysed with ACK Lysis Buffer (Lonza, Walkersville, MD), and mononuclear cells (MCs) were recovered and frozen in CryoStor CS5 (BioLife Solutions, Bothell, WA) and stored in liquid nitrogen before evaluation. PBMCs were isolated from peripheral blood collected in EDTA tubes using previously described standard methods (38 (link)), frozen in CryoStor CS5, and stored in liquid nitrogen prior to evaluation. PBMC samples taken on the day of pregnancy termination were used for most cases. If such a sample was not available, the closest available PBMC sample, prior to surgical pregnancy termination, was used. Full details on the PBMC samples used can be found in Supplementary Table 1.