Multi tissue dissociation kit 1

Human submandibular gland samples were placed in a C tube (Miltenyi Biotec, North Rhine-Westphalia, German) containing digestion buffer (Multi Tissue Dissociation Kit 1, Miltenyi Biotec), and tissues were dissociated by a gentleMACS™ octo dissociator with heaters (Miltenyi Biotec). Submandibular gland cells in cell suspension were separated using Percoll solution (GE Healthcare, Chicago, IL). Subsequently, cells were stained with anti-human CD45 PE (BD biosciences, Franklin Lakes, NJ) antibodies. Non-immune cells (CD45-) were purified from stained cells using a BD Aria III (BD).

Single-cell suspensions of various skeletal muscles (quadriceps, rectus abdominis and diaphragm) were obtained by digesting the tissues using the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, Auburn, CA, USA) according to kit instructions. The cells were passed through a 70 µM cell strainer, erythrocytes were lysed using 1× BD Pharm Lyse™ (BD Biosciences, Franklin Lakes, NJ, USA) and washed with autoMACS^® Running Buffer (Miltenyi Biotec, USA). Fc receptors were blocked with CD16/CD32 (BD Biosciences, USA) for 10 min. Extracellular surface markers were stained with an antibody cocktail of the Live/Dead stain, APC-Cy™7 anti-mouse CD45 (clone 30-F11, BD Biosciences, USA), APC anti-mouse Ly-6G (clone 1A8, BD Biosciences, USA), PE anti-mouse F4/80 (clone T45-2342, BD Biosciences, USA), FITC anti-mouse CD86 (clone GL-1, BioLegend, San Diego, CA, USA) each at a 1:100 dilution for 30 min on ice. For intracellular staining, the cells were fixed with Leucoperm™ Reagent A 10 min, washed and following the cells were permeabilized with Leucoperm™ reagent B (Bio-Rad, Hercules, CA, USA) containing BV421™ anti-mouse CD206 (clone C068C2, BioLegend, USA) at a 1:100 dilution. The cells were washed and then analyzed on the BD FACSVerse™ Flow Cytometer (BD Biosciences, USA). Data analysis was performed using FlowJo 7.6 software (FlowJo, Ashland, OR, USA) and normalized to tissue weight.

Lung tissue from biopsies kept in HypoThermosol was minced finely with scissors, placed in Multi Tissue Dissociation Kit 1 solution as recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany), and incubated at 37°C for 45 min in gentle agitation. The minced preparation was crushed on nylon mesh (1 mm) and filtered through successive nylon filters (500 µm, 100 µm, 40 µm). The cell suspension was washed in PBS (470 g, 15 min), processed to erythrocyte lysis, resuspended in RPMI + 2% FCS, filtered twice on 40 µm and counted by 3 independent measurements with a counting Malassez chamber slide. Trypan blue staining indicated that over 90% cells were viable.

Mice were subcutaneously immunized at the tail base with 50 μg of MOG_35–55 peptide (GenScript, Piscataway, NJ, USA) emulsified in complete Freund’s adjuvant (BD Biosciences). In addition, mice were intraperitoneally injected with 200 ng of pertussis toxin (List Biological Labs) on day 0 and day 2 post-immunization. Mice were observed daily to monitor body weight and EAE clinical symptoms on a scale from 0 to 5 as follows: 0—no disease; 1—flaccid tail; 2—impaired righting reflex and hind legs weakness; 3—complete hind legs paralysis; 4—complete paralysis of both hind legs with partial foreleg paralysis; and 5—moribund. To isolate cells from the spinal cord, the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec) was used as recommended by the manufacturer. The cells were processed as described in the protocol and subsequently subjected to flow cytometric analysis.

Fresh tissue dissociation and cryopreservation conditions were optimized to maximize cell viability and facilitate proper preservation of diverse cell types. Specifically, digestion enzyme titration was performed and freezing media were compared based on epithelial cell retention measured by flow cytometry after dissociation and freezing/thawing, respectively. The tissues were collected at a ~2 cm³ size and put in MACS Tissue Storage Solution (Miltenyl biotec cat.130-100-008) within 30 mins of surgical dissection. Samples were kept at 4°C and processed within 3 hours of dissection. Tissues were chopped into 2 mm diameter pieces within a dissociation tube to reduce cell loss. Multi Tissue Dissociation Kit 1 and gentleMACS Octo Dissociator (Miltenyi Biotec) were used for dissociation of the tissue into single-cell suspension with a reduced amount of enzyme R (25% of the regular amount). Red blood cells were removed using Red Blood Cell Lysis Solution (Miltenyl biotec cat.130-094-183). Single-cell suspension of samples was frozen using 90% FBS and 10% DMSO and stored in liquid nitrogen until further processing except for the time of transfer in dry ice.

Single cell suspensions were prepared from the bone marrow and spleen, followed by red blood cell (RBC) lysis by using RBC Lysis Buffer (BioLegend) for 5 min. For the flow cytometric analysis of peripheral blood, leukocytes from the heparinized blood sample were isolated from the interphase of 25% and 65% Percoll PLUS (cytiva) after gradient centrifugation. For the flow cytometric analysis of lung and skin samples, tissues were minced by razors and dissociated by using gentleMACS dissociator (Miltenyi Biotec) and Lung Dissociation Kit (Miltenyi Biotec, catalog #: 130-095-927) or Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, catalog #: 130-110-201). Cells were stained with the indicated antibodies after treatment with normal rat serum (Merck Millipore) and TrueStain FcX PLUS (2.5 μg/mL; BioLegend; clone: S17011E, catalog#: 156604, dilution 1:200) to prevent non-specific binding. Stained cells were analyzed with FACSLyric (BD Biosciences) and FlowJo software ver 10.8.1 (BD Biosciences) or sorted with FACS AriaIII (BD Biosciences). For ex vivo EdU uptake assay, cells collected from the bone marrow or lung were cultured for 2 h with 10 μM EdU (5-ethynyl-2′-deoxyuridine). After culture, EdU staining was performed by using Click-iT Plus EdU flow cytometry assay kits (ThermoFisher Scientific, catalog #: C10633) according to the manufacturer’s protocol.