Cytofunnel

Following FACS, separated CD26⁺ and CD26⁻ fibroblasts were incubated for 20 min in suspension with CD26-PE antibody (clone M-A261, 1:20, BD Biosciences, Allschwil, Switzerland). Subsequently, cell suspensions were washed in DBPS, fixed in 4% paraformaldehyde, resuspended in DPBS, and cytocentrifuged using cytofunnels (Thermo Fisher Scientific, Basel, Switzerland) attached to glass slides. Stained cells were visualized using a Nikon Eclipse TE2000-U inverted fluorescent microscope (Nikon, Tokio, Japan).

To attach isolated satellite cells on glass slides for cell size analysis and immunostaining, we put cells on cytofunnels (5991040, Thermo Fisher Scientific, Waltham, MA), assembled them with slide glasses filter cards, and spun down at 1,300 rpm for 3 min (Cytospin 3, Shandon). After centrifugation, cells were fixed with 2% paraformaldehyde for 10 min and washed with PBS 3 times before immunostaining.

The number of live cells in the PEC lavage samples was counted with a hemocytometer as follows. An aliquot of 150 μL of the cell suspension was transferred to a Cytofunnel (ThermoFisher, Waltham, MA, USA) and centrifuged at 10xg in a Shandon Cytospin 4 centrifuge for 5 min for deposition onto a glass slide (ThermoFisher). The slides were dried and stained using a Diff-Quick kit following the manufacturer’s protocol (RAL Diagnostics, Martillac, France). Cells were phenotyped by microscopy at 100X with oil-immersion. The slides were incubated for 24 and 48 hours at 37°C, dried and stained using a Diff-Quick kit to visualize phagocytosis of L. major by macrophages and neutrophils. The remaining volumes of the PEC lavage samples were centrifuged at 297xg for 10 min to separate cells and supernatants. Supernatants were aliquoted and frozen at -80°C for further analyses. Cell pellets were resuspended in 1mL Trizol (ThermoFisher) for 5 min and frozen at -80°C for further analyses.

Following methods previously described (83 ), the lungs of sacrificed mice were first inflated with 1 ml of PBS with 0.1 mM EDTA using a tracheal catheter. About 1 ml of BAL was then collected and centrifuged at 1500 rpm. The supernatants were stored for subsequent ELISA experiments at −80 °C. Ammonium–chloride–potassium buffer was then added to the cell pellet and incubated on ice for 5 min. After centrifuging the sample again, the cell pellet was resuspended with 500 μl of RPMI medium. Total BAL cell counts were recorded using a hemocytometer. For differential cell counts, 50,000 cells were loaded into a Cytofunnel (Thermo Fisher Scientific) and centrifuged for 10 min at 600 rpm. Slides were then stained using Diff-Quick stain kit. Differential cell counts were recorded based on morphology and staining patterns.
Using an ELISA kit (Thermo Fisher Scientific), mouse IgE was measured from supernatants of BAL following the manufacturer’s protocol. These measurements were normalized to total protein in these supernatants, which was measured using the Pierce Rapid Gold BCA Protein Assay.

Non-cryopreserved or cryopreserved cells were fixed and stained with FITC-conjugated anti-CD63 and AF647-conjugated anti-Granzyme B antibodies. Cells were then washed twice and loaded onto EZ Double Cytofunnel (Fisher Scientific #A78710005) with glass slides. Cytofunnel was spun down to attach cells to the glass slides at 600 RPM for 10 min using mounting media ProLong Gold Antifade Reagent DAPI (ThermoFisher # P36931).