T rk s solution

3 × 10⁷ cells of the final Treg product were transferred to a 15 ml tube and centrifuged (300 g, 10 min, RT). The supernatant was discarded and the cell pellet was lysed in 2 ml of distilled water, vortexed and incubated for 5 min at 37°C. Subsequently, 200 μl of a 2,000 U/ml DNase I stock solution (Roche) were added and the sample was incubated for 2 min at RT. After incubation, 6 ml of MACSQuant Washing Solution (Miltenyi Biotec) were added and the vial was incubated for 10 min at RT. After centrifugation at 1,400 g for 10 min at RT, the supernatant was carefully removed and the residual beads were resuspended in the remaining volume (<30 μl). Residuals beads were quantified using a C-Chip™ Neubauer improved disposable counting chamber (Biochrom AG) and Türk's solution (Merck Millipore) at a 1:1 dilution ratio.

For the toxicity assessment of GTN057 in vivo, we intraperitoneally (i.p.)‐injected male 5‐week‐old ICR mice (CLEA, Tokyo) with GTN057 (0, 50, or 100 mg/kg) dissolved in 1% Tween® 80 + 10% DMSO in saline on 2 consecutive days of every 3‐day period for 2 weeks. The mice were weighed every week. Peripheral blood was collected with a heparinized hematocrit tube (Terumo, Tokyo) from the tail vein of each mouse every week and was stained with Türk's solution (Merck). The numbers of leukocytes and neutrophils were counted using light microscopy.

Migration capacity of isolated PMNL was examined using modified Transwell 24-well chambers (Corning, New York, USA) with 3-μm pores. Before the evaluation of the migratory rate of neutrophils, cell number was equalized to 1 × 10⁶ vital cells/ml. Subsequently, 100 μl of the cell suspension were placed in the upper chamber. The lower chamber contained 500 μl RPMI 1640 with supplements and either IL-8 (10 ng/ml, R&D Systems), the recombinant human CC16 (CC16_I: 100 ng/ml, CC16_II: 33.33 ng/ml and CC16_III: 1 ng/ml, respectively, R&D Systems) or 20% serum obtained from TP. The sera from TP were either pre-incubated with or without anti-CC16-antibody for CC16-neutralization (10 ng/ml, human uteroglobin affinity purified Ab, R&D Systems). After incubation for 3 h at 37 °C and 5% CO₂, the upper transwell chamber was removed and cells migrating to the lower chamber were collected and counted after their staining with Türks-solution (1: 1, Merck). The number of cells migrating into the lower compartment was quantified. The migratory capacity of control (ctrl) cells that were incubated without serum in the lower compartment was set as 100%.

For measurement of the blood parameters, mice were CO₂-euthanized, and blood was drawn from the retrobulbar plexus. Hematocrit was determined by taking blood with hematocrit capillary (BRAND GMBH + CO KG, Wertheim, Germany) at the retrobulbar plexus and then centrifuged with 11,500× g for eight minutes at room temperature (RT) [38 ].
The number of erythrocytes (RBC) and leukocytes (WBC) were determined by filling specific pipettes with blood, and 0.9% NaCl solution (RBC) or “Türk’s solution” (Merck KGaA, Darmstadt, Germany) (WBC) was added. Cells were counted by using a “Neubauer improved” counting chamber, and a 40× objective (RBC) or a 10× objective (WBC) of the microscope was used. Finally, the concentration of RBC or WBC was calculated according to the literature [38 ].
Hemoglobin capillary (BRAND GMBH + Co KG, Wertheim, Germany) was filled with 20 µL of blood, added to 5 mL of the “transformation solution” (0.02% K₃Fe(CN)₆, 0.005% KCN, 0.014% KH₂PO₄ in ddH₂O) and incubated for 15 min at RT. The extinction of the solution was measured against the “transformation solution” (λ = 546 nm), and the concentration of hemoglobin was calculated [38 ].
MCH and MCV were calculated with the blood parameters measured before [38 ].

Eight-day-old rats were injected intraperitoneally with 1 mg/kg PAM3CSK4 (P3C, Merck Millipore France), or 0.9% saline. CSF was sampled 14 h later [5 (link)], and total leukocytes and polymorphonuclear neutrophiles (PMNs) were counted in a Bürker chamber after staining with Türk’s solution (Sigma-Aldrich). The phenotypic analysis of whole blood immune cells was performed by immunocytochemistry following red blood cell lysis as previously described [26 ].