Cell dissociation buffer enzyme free pbs based

The Polydimethylsiloxane (PDMS)-based microfluidic device was fabricated as previously described [99 (link), 100 (link)]. The PDMS-based microfluidic devices contained a series of parallel microchannels with varying widths of 3, 6, 10, 20, and 50-µm, lengths of 200 µm, and heights of 10 µm. The microchannels were perpendicular to a 2D cell seeding area and were coated with 20 µg/ml of collagen type I at 37 °C for 60 to 80 min. Cells were dissociated with PBS-Based Enzyme Free Cell Dissociation Buffer (Gibco). 1,000,000 cells per mL of media were loaded into the microfluidic device. Cells were imaged via live-cell time lapse microscopy using the EVOS FL Auto Imaging System (Life Technologies). Images were taken with a 10X objective every 30 min for 24 h. Environment chamber conditions were 37 degrees Celsius, 5% CO2, and 20% O2. 4

Tetraploid embryos were generated by heat-shock treatment, following the HS2 protocol adapted from⁴⁴ . Embryos were collected 2 min post fertilization and maintained in E3 media for a further 20 min at 28 °C, during which microinjection of MOs was performed. Embryos were heat-shocked between 20 and 23 min post fertilization by incubation in E3 media at 42 °C for 2 min, then returned to E3 media at 28 °C and allowed to develop normally. Embryos were monitored at the normal point of the second cell division (1 h post fertilisation (hpf)) and heat-shock treated embryos that failed to undergo cytokinesis were selected for downstream analysis, along with matched non-heat-shock treated controls. For the assessment of ploidy manipulation, embryos were collected at the 12-somite stage, dissociated with PBS-based enzyme-free cell dissociation buffer (Gibco), washed with PBS and subjected to propidium iodide DNA content analysis following manufacturer’s conditions (Invitrogen). Flow cytometry was performed on the CyAn ADP machine (Beckman Coulter) using the Summit 4.4 software package using the standard gating strategy.

Our lab has recapitulated published protocols for the generation of mature myeloid cells from an induced pluripotent stem cell (iPSC) source (Yanagimachi et al. 2013). Briefly, iPSCs are transformed in four culture steps to become floating CD14⁺ myeloid cells. CD14 cells are isolated from the supernatant using human CD14 Microbeads (Miltenyi Biotec) and magnetic columns. CD14 cells are then cultured for 7 days in 10% FBS 1640 media supplemented with GMCSF (50 ng/mL; Miltenyi Biotec) to generate resting myeloid cells (M0). We harvest M0 cells with enzyme-free/PBS-based cell dissociation buffer (Gibco) and plated at 5 × 10⁴ cells per well in a 24-well plate. M0 cells are allowed to settle for an hour followed by a stimulation using lipopolysaccharide (LPS) (0.1 ng/mL; Sigma) and gamma interferon (IFN-γ) (0.2 ng/mL; eBioscience) to create activated, pro-inflammatory myeloid cells (M1). Tregs are co-cultured at 1:1 ratio with M1’s overnight (~20 h) to assess suppression of myeloid-specific pro-inflammatory markers. Cultured Tregs and M1 cells are individually isolated for RNA transcript analysis and cultured media collected to assess cytokine protein levels via ELISA (Sigma).

Bone marrow cells were flushed out of the long bones of the mice and the cell suspension cultured in αMEM supplemented with 10% FCS, Pen/Strep and 100 ng/ml M-CSF (Prospec Bio) for three days. Next the non-adherent cells were removed by washing the cultures with PBS, and the adherent BMDMs harvested using Acutase (Sigma) or cell dissociation buffer (Enzyme-Free; PBS-based) (Gibco, Life Technologies). For osteoclast formation experiments, cells were plated in 96-well plates at 15 × 10³ cells in 100 µl medium supplemented with 25 ng/ml M-CSF and up to 100 ng/ml RANKL (a kind gift of Dr. J. Dunford, University of Oxford) per well. The culture medium was refreshed at day 2 and day 4 and the cells fixed on day 5 with 4% buffered formalin and stained for TRAP. TRAP positive cells containing three or more nuclei were counted as osteoclasts. For RNA isolation, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF only (for macrophage cultures) or 25 ng/ml M-CSF plus 100 ng/ml RANKL (for osteoclast cultures) and cultured for 5 days as described above. For Western Blot analysis, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF and cultured for 3 days.