Ghost red

WEHI-231 cells (PRL receptor⁺) and immature B-cells from MRL/lpr mice were incubated with PRL (50 ng/mL) for 1 hour before stimulating the cells with anti-IgM F(ab′)₂ (10 μg/mL) for 48 hours and 18 hours, respectively. Cells cultured with medium, PRL, or anti-IgM F(ab′)₂ were used as controls. The cells were washed with PBS and incubated with Ghost-Red (Tonbo Biosciences, California, USA) for 30 minutes at 4°C; Ghost-Red was used to measure viability (live cells do not stain and remain Ghost-Red⁻). Data were acquired using a MACSQuant Analyzer 10 cytometer (Miltenyi Biotec) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

For BAL fluid collected, leukocyte populations were gated as previously described (33 (link)). Single cell suspensions of lung cells were stained for ILC2 as Live CD45⁺ Lineage⁻ Thy1-2⁺ CD127⁺ ST2⁺. For cytokine detection cells were stimulated for 4 h with PMA Ionomycin and Brefeldin A (cell stimulation cocktail, Biolegend) or BrefeldinA alone. After 15 min of viability stain with GhostRED (Tonbo, San Diego CA), cell fixation and intracellular cytokine staining was performed according to manufacturer instructions (BD biosciences San Jose CA) using IL-5-BV421, IL-13-APC, IL-17a-PeCy7 (Biologend, San Diego CA). For Transcription factor staining, FOXP3 fixation permeabilization kit was used according to manufacture and stained with GATA3-PECy7, ki67-e450, and IRF4-PE (eBioscience San Diego, CA). For mitochondrial analysis, ILC2 cultured with 20 nM MitoTrackerGreen and 100 nM MitoTrackercmxROS-H₂ or 5 uM mitoSOX (Invitrogen, Carlsbad CA) in PBS with GhostRED dye for 30 min at 37°C 5% CO₂. Cells were acquired on BD Canto II and analyzed with FlowJo software (Treestar Ashland OR).

WEHI-231 cells and immature B cells from 9-week-old mice were preincubated for 30 min with G129R (PRL-inh), GSK, or Stattic and incubated with PRL for 1 h before stimulating them with anti-IgM F(ab)2 (10 μg/mL) to induce clonal deletion or apoptosis for 48 h (WEHI-231) or 18 h (murine B cells). Cells were washed with PBS and incubated with Ghost-Red (Tonbo Biosciences, San Diego, CA, USA) at 4 °C for 30 min. For caspase-3 staining, the cells were permeabilized with Cytofix or Cytoperm (BD Biosciences) at 4 °C for 1 h, washed with Perm/wash, and incubated with anti-caspase-3-FITC at 4 °C for 1 h. Data were acquired using a MACSQuant Analyzer 10 cytometer and analyzed with FlowJo software. Cells cultured in a medium without inhibitors and anti-IgM F(ab)2 were used to compare PRL-induced apoptosis.