Countess fl automated cell counter

Stem cells were transplanted immediately after the removal of the embolectomy catheter. In the case of transplantation of fresh MSCs, the cells were cultured in a 150 mm culture dish until they had reached 90% confluence. The cells were harvested, centrifuged, and pelleted as described above. In the case of transplantation of frozen thawed MSCs, the cryopreserved cells were thawed by warming the cryovial at 37°C for 5 min. The cell suspension was mixed with an equal volume of DMEM and centrifuged at 2,500 rpm for 5 min at 4°C. The medium was discarded and the cell pellet was resuspended in PBS and centrifuged. The cell pellets obtained from both fresh and frozen thawed preparations were resuspended in 1 mL of Hartmann's solution. The cell number required for transplantation was counted using a Countess FL Automated Cell Counter (Thermo Fisher Scientific, Pittsburg, PA, U.S.A.) after staining with Trypan blue. The %age of GFP expression for HO-1 MSCs, GFP MSCs, and FT-HO-1 MSCs was 91.03 ± 0.8, 91.30 ± 0.7, and 90.56 ± 0.95, respectively. About 1 × 10⁷ fresh GFP MSCs and HO-1 MSCs and 1.5 × 10⁷ frozen thawed HO-1 MSCs were diluted in 20 mL of Hartmann's solution and injected by slow IV infusion in 10 min. The cells were injected IV for three consecutive days with an interval of 24 h between each injection. The dogs were kept for four weeks after cell transplantation.

Cell viability was assessed using the Countess FL Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA). The semi-automated cell count was performed using the manufacturer’s instructions. The cell sample (10 μL) was mixed with 10 μL of 0.8% trypan blue stain (Sigma Aldrich, St. Louis, MO, USA). The view range and properties were optimized according to manufacturer’s instructions and the final results was confirmed with the post-analysis of the view screens. This method was validated for performing cell viability assessment [63 (link)]. The viability was assessed before each experiment as part of standard protocol and cell counting. We proceeded with the experiment if the cell viability was ≥95% in both cultivation conditions.

Cell viability was assessed using the Countess FL Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA), an automated technique which has been validated for assessing cell viability [71 (link)]. The viability was assessed before each experiment as part of standard protocol and cell counting. We proceeded with the experiment if the cell viability was ≥95% in both cultivation conditions.

NSPCs were cultured in monolayer for five days until 80–90% confluency was attained, and passaged into three 60 mm PLO/fibronectin-coated petri dishes. At each passage, three plate cells were counted and viability assessments were performed using a Countess FL Automated Cell Counter (Thermo Fisher Scientific, Pittsburg, PA, USA) which is using trypan blue staining. The population doubling level (PDL), a cumulative counting technique, was applied using the following formula: PDL_(n/n−1) = log (N_f/N₀)/log 2, where n = passage number, N_f = final number of cells, and N₀ = number of cells seeded at passage. To obtain the new PDL(n + 1), PDL(n/n − 1) was added to the previous PDL(n) [53 (link)].
For growth curve analysis, cells at the 3rd, 6th, and 9th passage were cultured in 24-well plates (coated 12-mm cover slips were inserted into each well). Cell counts were obtained daily for seven days using trypan blue to derive a weekly growth curve.