Hemocytometer

To induce pneumonia, mice were inoculated with viable PAK, PAKflic (5 × 10⁶ CFU) or K. pneumoniae (1 × 10⁴ CFU) or flagellin purified from P. aeruginosa (1 μg, tlrl-pafla, Invivogen) intranasally. At predefined time points mice were euthanized by heart puncture after injection of ketamine/medotomidine as described [59 (link),64 (link)]. Briefly, the right lung was used for bronchoalveolar lavage (BAL) by instilling 2 × 0.5 ml of sterile phosphate-buffered saline; the left lung was preserved for histopathology after fixation in 10% formalin. Bronchial brushing was performed after BAL to collect bronchial epithelial cells as described [40 (link)]; BALF was serially diluted and plated on blood agar plates for measurements of bacterial numbers. Cell counts in BALF were determined using a hemocytometer (Beckman Coulter, Fullerton, CA). BALF supernatants and bronchial brushes were stored at −20°C until further analysis. In some experiments BALF was pelleted and cells were lysed for RNA isolation. For all animal experiments, littermate controls and conditional knockout were mixed in cages (i.e. randomized) and procedures were done without knowledge of the genotype (blinded). Readout parameters were thereby determined in a double-blinded manner.

Platelets were obtained from blood samples [in presence of acid citrate dextrose (ACD) as anticoagulant] of healthy donors, that signed informed consent from Transfusional Center of Policlinico Umberto I, Sapienza University of Rome.
Platelet-rich plasma (PRP) was preliminary separated from the whole blood by centrifugation at 150 x g for 15 min at 20°C. Two thirds of the PRP, with the addition of ACD, to prevent platelet activation, were transferred into a new another sterile tube, without disturbing the buffy coat layer, in order to avoid contamination. PRP was centrifuged at 900 x g for 10 min at 20°C (with no brake applied). Platelet-poor plasma (PPP) was discarded and platelet pellets were resuspended in Tyrode’s buffer, containing 10% (v:v) ACD. Then, after washing, as above, platelets pellets were resuspended in Tyrode’s buffer, containing Bovine Serum Albumin (BSA, 3 mg/ml).
Platelet were counted by a hemocytometer (Coulter, Beckman Coulter, Brea, California, USA), which gives that leukocyte contamination was < 1 leukocyte/10⁷ platelets. The purity of the isolated platelets was analyzed and confirmed by staining with a fluorescein isothiocyanate (FITC)–conjugated anti-CD41 antibody (Beckman Coulter) and using flow cytometry (Coulter Epics, Beckman Coulter; data not shown).

JHH-6, JHH-7, Kami41, PLC/PRF/5, JHH-7 and HuH-7 cell lines were suspended in serum-free medium containing 0.5% fetal bovine serum, seeded into a 24-well culture plate (5 × 10³ cells/well; TPP Techno Plastic Products AG, Trasadingen, Switzerland) and incubated for 7 days at 37 °C in 5% CO₂. Additionally, the JHH6 and JHH7 cell lines were further treated with either DMSO or MK2206 (Sigma) at the indicated concentrations and incubated for 7 days at 37 °C in 5% CO₂. The number of living cells was counted using either a hemocytometer or Coulter Counter (Beckman Coulter, Inc., Pasadena, CA, USA). Transwell migration assays were performed as described previously [45 (link)]. Briefly, transwell inserts with 8 μm-sized filters (Falcon) were inserted into 24-well plates. Growth medium containing 10% FBS were added to the lower chamber, while a cell suspension (2 × 10⁴ cells) in growth medium containing 0.5% FBS was introduced into the upper chamber. The plates were incubated at 37 °C in 5% CO₂ for 4 h. After incubation, the cells that had migrated to the lower side were stained with 0.25% crystal violet/20% methanol solution and counted using a light microscope at ×10 magnification. The values obtained represent the averages of the five fields.

Platelets were prepared from blood samples of healthy donors, with the addition of acid citrate dextrose (ACD) as anticoagulant. Platelet-rich plasma (PRP) was preliminary obtained from the whole blood by centrifugation at 150 × g for 15 min at 20°C.
PRP was centrifuged at 900 × g for 10 min at 20°C. Platelet-poor plasma (PPP) was removed and pellets were re-suspended in Tyrode's buffer, containing 10% (v:v) ACD. Then, after washing, pellets were re-suspended in Tyrode's buffer, containing Bovine Serum Albumine (BSA), 3 mg/ml.
Platelet counts were performed by a hemocytometer (Coulter, Beckman Coulter, Brea, California, USA); that leukocyte contamination was <1 leukocyte/10⁷ platelets. The purity of the isolated platelets was confirmed by staining with a fluorescein isothiocyanate (FITC)–conjugated anti-CD41 mAb (Beckman Coulter) and analyzing by flow cytometry (Coulter Epics, Beckman Coulter).

For each sample, 1 mL pre- and post-SECCS bone marrow was treated with red blood cell lysis buffer (BioTime, Shanghai, China). Then, the nucleated cells (NCs) were counted using a hemocytometer (Beckman Coulter, Brea, California, USA). Cell viability was assessed by the trypan blue exclusion rate (Vi-CELL XR Cell Viability Analyzer Software, Beckman Coulter). The difference between pre- and post-SECCS bone marrow was estimated for each patient.

To obtain the BALF, the lungs were lavaged three times with ice-cold PBS (0.5 ml) and withdrawn each time using a tracheal cannula (a total volume of 1.5 ml). The collected BALF was centrifuged at 3000×g for 10 min at 4 °C and the supernatant was collected and frozen at − 80 °C for subsequent assays. The cell pellet was resuspended in PBS, and after excluding dead cells by trypan blue staining, the total inflammatory cells in BALF were determined by counting cells with a hemocytometer (Beckman Coulter, Inc). In order to analyze differential cell counting, 100 μl of BALF was centrifuged onto slides by a Cytospin (Thermo Fisher Scientific, Waltham, USA). After the slides were dried, the cells were fixed and stained using Wright Stain solution (32857, Sigma-Aldrich, USA) according to the manufacturer’s instructions. The number of polymorphonuclear neutrophils (PMNs) was classified by a laboratory technologist blinded to the experiment, to obtain the percentage of neutrophils. The frozen BALF supernatant was thawed and thoroughly mixed; then, total protein concentration was determined by BCA (bicinchoninic acid) method.

To assess proliferation, cells were counted daily using a hemocytometer or Z1 Beckman Coulter counter before and after rapamycin induction. For the clonogenic survival assay, cells were induced for 72 h with rapamycin or DMSO, counted and diluted to 1.6 parasites mL⁻¹ in HOMEM with 20% heat-inactivated FCS and 100 nM rapamycin or DMSO. The cells were plated out in two to four 96-well flat bottom plates by adding 200 µL cells per well. Plates were sealed and incubated at 25 °C for 3–4 weeks. Surviving clones were counted by visual inspection of each well using a light microscope; any well containing live parasites was counted as a surviving clone. The percentage of surviving clones of the total cells plated is shown in graphs.