Z2 coulter particle count and size analyzer

Automated white blood cell (WBC) counts (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter Inc., FL, USA) and manual differential counts were performed (300 cells total) on heparinized blood samples collected at 0, 2, 6 and 19 dpi. PBMC were isolated by gradient centrifugation (Ficoll-Paque™ PLUS, GE Healthcare, Mississauga, ON, Canada) and analyzed phenotypically by flow cytometry (FCM) as described in detail [17 (link)]. Major PBMC populations were defined as follows: myeloid cells (CD172a⁺), natural killer (NK) cells (CD8α⁺CD3⁻), B cells (CD79α⁺), γδ T cells (T cell receptor γδ⁺), T helper cells (CD3⁺CD4⁺), and cytolytic T cells (CTLs) (CD3⁺CD8β⁺). Absolute numbers of different cell subsets were calculated using results from automated WBC and differential counts (total number of lymphocytes plus total number of monocytes).

Chlamydomonas reinhardtii UVM4 cells (Neupert et al., 2009 (link)) were grown in Tris-Acetate-Phosphate (TAP) medium (Kropat et al., 2011 (link)) on a rotatory shaker. For transformation, cells were grown at a light intensity of 100 μmol photons m^–2 s^–1 to a density of 5 × 10⁶ cells/ml and collected by centrifugation at 4000 × g for 2 min. 5 × 10⁷ cells were mixed with 1 μg DNA linearized with NotI and transformed by vortexing with glass beads (Kindle, 1990 (link)). Vortexed cells were diluted twofold with TAP and 2.5 × 10⁷ cells were spread onto TAP agar plates containing 100 μg ml^–1 spectinomycin. Plates were incubated over-night in the dark and then incubated at 30 μmol photons m^–2 s^–1 for about 10 days. For growth curves, cells were inoculated in 100 ml TAP medium and grown at 150 μmol photons m^–2 s^–1 to a density of about 8 × 10⁶ cells/ml. 100 ml TAP or Hepes-Minimal-Phosphate (HMP) medium (5 mM Hepes-KOH instead of 20 mM Tris, no acetate) were then inoculated with 3 × 10⁵ cells/ml in triplicates for each strain and growth was monitored by cell counting using the Z2 Coulter Particle Count and Size Analyzer (Beckmann). The culture volume is the summed cell volume of all cells in 1 ml medium. For mass spectrometry analyses, samples were harvested 22 h after inoculation (early log phase).

The rats in the study were euthanized by cervical dislocation during the fourth week after injection. Their BMSCs were obtained by flushing the bone marrow from their tibias and femur bones using low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, USA), 100 U/ml streptomycin, 100 U/ml penicillin and 200 U/ml heparin (Sigma-Aldrich, St Louis, MO), according to previously described procedures [29 (link),30 (link)]. The primary cells were cultured in low-glucose DMEM supplemented with 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin at 37°C in a 5% CO₂ atmosphere. Non-adherent cells were discarded via a change of medium after 24 hours, and the medium was replaced every three days until the cells reached 80–90% confluence (S1 Fig). A Z2 Coulter particle count and size analyzer (Beckman Coulter, USA) was used to evaluate the quantity of cells. The quantities of both normal and diabetic BMSCs were approximately 1.2–1.5×10⁷ cells/dish. Next, 0.25% trypsin (EDTA) was used for cell detachment, and the BMSCs were subcultured at a density of 1×10⁵ cells/ml. Cells at passage 2 or 3 were used for the subsequent experiments. Osteogenic medium (DMEM, 10% FBS, 10 mM dexamethasone, 50 μg/ml L-2-ascorbic acid and 10 mM glycerophosphate) was used to induce the osteogenic differentiation of BMSCs.

HCT116 DNA-PK_cs +/+, +/−, −/−, or KD/− cells were seeded (3 × 10³) in each well of a 6-well tissue culture dish. The increase in cell number was determined by counting via Beckman Coulter Z2 Coulter^® Particle Count and Size Analyzer at daily intervals starting at Day 4 and ending on Day 7 post-plating. The results are presented as Mean ± SD, from three biological repeats with a P-value that was obtained using a Student t-test.

Porcine alveolar macrophages (AMac) were isolated from healthy conventional pigs as previously described [53 (link)]. Briefly, lungs were lavaged with 300 ml of PBS. Collected lavage was centrifuged at 400 × g for 15 min, cells washed once with PBS, and cells resuspended in supplemented medium [RPMI 1640, 5% swine sera, 5 mM HEPES, 1 mM L-glutamine, antibiotic-antimycotic, and 50 μg/ml gentamicin (Invitrogen Life Technologies)]. Cells were cultured in 150 × 15-mm petri dishes for 2 h at 37°C in 5% CO₂. Non-adherent cells were removed and adherent AMac harvested with a cell scraper, collected, washed once, and counted on a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter). AMac were seeded at 2.5 × 10⁵ cells per well in a 48-well flat-bottom plate with a final volume of 0.5 ml for studies (RT-PCR and cytotoxicity). Cells were allowed to adhere for an additional 2 hours before stimulation. Non-stimulated cells were included for each biological sample as a control.

Automated white blood cell (WBC) counts (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter Inc., FL, US) and manual differential counts were performed (300 cells total) on heparinized blood samples collected on D0, D2, D6 and D19. Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare, Mississauga, ON) and analysed phenotypically by flow cytometry (FCM). Major PBMC populations were defined as follows: myeloid cells (CD172a⁺), NK cells (CD8α⁺CD3^-), B cells (CD79α⁺), and T cells (CD3⁺). In addition, three distinct subpopulations of T cells were identified and analyzed: γδ T cells (T cell receptor γδ⁺), T helper cells (CD3⁺CD4⁺), and cytolytic T cells (CTLs) (CD3⁺CD8β⁺). Absolute numbers of different cell subsets were calculated using results from automated WBC and differential counts (total number of lymphocytes plus total number of monocytes).