6.03 Calorimetry Lab
1. The final yield of the cells can be determined by use of an automated cell counter or by use of a hemocytometer (Neubauer chamber). In our laboratory, we determine the number of purified cells in a Neubauer-improved, bright line chamber with V-slash with a depth of 0.1 mm and a counting area of 0.0025 mm2 from Marienfeld (Lauda Königshofen, Germany, PN # 0650030).
2. For determination of the final cell yield, mix a 10 µl aliquot of the purified cells (Figure 9D) with 10 µl of trypan blue solution (Figure 9E).
3. Place the glass cover on the top of the Neubauer chamber so that it covers the central area. Note: in this step it is essential that the glass cover is tightly sticking to the Neubauer chamber. To guarantee this we slightly heat …show more content…
Load the counting chamber with 10 µl from the solution prepared in 2 (Figure 9F). Note: Please avoid introducing air bubbles into the counting chamber.
5. Place the Neubauer chamber on the microscope stage (Figure 9G) and count the cells in a big square (which is equal to 12 small squares). The typical appearances (i. e. high content of fat and rounded cell phenotype) of four cells in a small square of the counting chamber are given in Figure …show more content…
Remove the DAPI solution and wash the cells with ice cold HBSS (without Ca/Mg).
9. Mount the cells and document the results as described above (3.8.1, steps 11-12).
3.9 Cell culturing
1. Dilute the purified cells (unsorted or sorted) in HSC culture medium and plate the cells at a density of 2 x 104 cells/cm2.
2. Incubate the cells in a humidified incubator with 5% CO2 at 37oC.
3. Immediately after cell attachment, replace the medium and remove the plates to the incubator.
4. Note: The culture process on uncoated plastic will lead to "transdifferentiation" in which the cells lose their quiescence resulting in an activated phenotype in which the cells slowly loose their vitamin droplets and acquire a typical star-shaped morphology. The activation of the cells is further acquired by elevated expression of α-SMA and collagen type I. After the cells reach confluence, the cells can be enzymatically detached from the plates by trypsin or accutase and subcultured for four passages. Please note that the proteolytic detachment of the primary cells further promotes the activation process that finally results in a myofibroblastic phenotype. Some representative images of isolated HSC that were grown in culture for prolonged times on uncoated plastic are shown in Figure
2. For determination of the final cell yield, mix a 10 µl aliquot of the purified cells (Figure 9D) with 10 µl of trypan blue solution (Figure 9E).
3. Place the glass cover on the top of the Neubauer chamber so that it covers the central area. Note: in this step it is essential that the glass cover is tightly sticking to the Neubauer chamber. To guarantee this we slightly heat …show more content…
Load the counting chamber with 10 µl from the solution prepared in 2 (Figure 9F). Note: Please avoid introducing air bubbles into the counting chamber.
5. Place the Neubauer chamber on the microscope stage (Figure 9G) and count the cells in a big square (which is equal to 12 small squares). The typical appearances (i. e. high content of fat and rounded cell phenotype) of four cells in a small square of the counting chamber are given in Figure …show more content…
Remove the DAPI solution and wash the cells with ice cold HBSS (without Ca/Mg).
9. Mount the cells and document the results as described above (3.8.1, steps 11-12).
3.9 Cell culturing
1. Dilute the purified cells (unsorted or sorted) in HSC culture medium and plate the cells at a density of 2 x 104 cells/cm2.
2. Incubate the cells in a humidified incubator with 5% CO2 at 37oC.
3. Immediately after cell attachment, replace the medium and remove the plates to the incubator.
4. Note: The culture process on uncoated plastic will lead to "transdifferentiation" in which the cells lose their quiescence resulting in an activated phenotype in which the cells slowly loose their vitamin droplets and acquire a typical star-shaped morphology. The activation of the cells is further acquired by elevated expression of α-SMA and collagen type I. After the cells reach confluence, the cells can be enzymatically detached from the plates by trypsin or accutase and subcultured for four passages. Please note that the proteolytic detachment of the primary cells further promotes the activation process that finally results in a myofibroblastic phenotype. Some representative images of isolated HSC that were grown in culture for prolonged times on uncoated plastic are shown in Figure