Trucount tube

Chemotaxis of individual peritoneal B cell populations in response to S1P was quantified using Transwell inserts (pore size 5 µm; Corning incorporated Costar) in a 24-well plate (Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany) according to the manufacturer’s recommendation. Absolute cell numbers of peritoneal B cell subsets were determined in an aliquot of the starting population by FACS analysis after staining with the antibody panels described above and using BD Trucount tubes (BD Biosciences). A total of 5 × 10⁵ peritoneal cells were added to the upper chamber in a volume of 100 µL. The lower chamber was filled with 600 µL migration medium containing different S1P concentrations (0–1000 nM). Cells were incubated for four hours at 37 °C and 5% CO₂. Migrated cells collected in the lower chamber and absolute cell numbers were determined by FACS after staining with the antibody panels described above and using BD Trucount tubes (BD Biosciences). To specifically block S1P₁, peritoneal lavage cells were pre-incubated with 1 µM Ex26 for 30 min at 37 °C and 5% CO₂ [45 (link)]. One µM Ex26 was added to the migration medium of the upper and lower chamber of the Transwell migration assay. Migration (%) was calculated as follows: (Absolute cell number of lower chamber/absolute cell number of inserted cells in upper chamber) × 100.

Blood sampling from the orbital sinus was performed for terminal blood collection by trained personnel, as described previously [11] . Briefly, wild-type (n = 180) or post-MI mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (6 mg/kg body weight) intraperitoneally and one orbital sinus was punctured using a glass pipette. Blood (0.8–1.0 ml) was collected into EDTA tubes and 20 or 50 µl were immediately transferred into BD TruCount tubes using reverse pipetting. For serial sampling, 20 µl of blood were obtained by a trained investigator via bleeding from the submandibular vein using a lancet (without anesthesia) as described previously [12] and immediately transferred into BD TruCount tubes for antibody staining.

The enumeration of HSPC was conducted in whole UCB using BD Trucount tubes (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instruction. 100 µl whole UCB were added by reverse pipetting to BD Trucount tubes and stained with anti- CD45− FITC, anti- CD34− PE, anti- CD38− PE- Cy7, anti- CD10− PE- CF594, 7-AAD (all BD Biosciences) and anti- CD133– APC (Miltenyi Biotechnology, Bergisch, Gladbach, Germany). After immunofluorescence staining, erythrocytes were lysed with ammonium chloride lysis solution and analyzed within one hour by flow cytometry (LSRII, BD Biosciences) and analysed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For enumeration of HSPC in UCB, CD34+ cells were gated according to the modified ISHAGE criteria (CD45^dim/7-AAD-/CD34+ cells). CD10 was added to exclude B- lymphoid progenitors. The number of cells/µl was calculated as [(# gated cells/# acquired beads) * (# of beads per test/test volume).

Complete peripheral blood was used to evaluate the lymphocyte subpopulations in healthy subjects and patients via flow cytometry. The blood sample (50 μL) assessed absolute cell counts using BD Multitest™ CD3/CD8/CD45/CD4, Becton Dickinson, USA and BD Trucount™ Tubes, Becton Dickinson, USA. Samples were incubated for 30 min at room temperature; erythrocytes were lysed using a LNW protocol. After staining, 2500 lymphocytes were acquired in a FACS Canto II flow cytometer, Becton Dickinson, San José California, USA, and were analyzed using FACS Canto software version 3.0, Becton Dickinson, San José, CA, USA.