Fig 1.
Summary of the experimental design of the somatic cell preparation and the various treatments applied, i.e. storage in milk or PBS for 72 h, variation of the centrifugation rates and heat treatment.
Table 1.
List of antibodies, fluorescence markers and isotype control for differentiation and labeling of bovine somatic cells applied on flow cytometry.
Fig 2.
Flow cytometry scatter pattern for the identification of differential somatic cells in blood-milk mix cell suspension (1/1, v/v) and corresponding isotype control sample, respectively.
(A) FSC/SSC dotplot of cell suspension. All somatic cells (in yellow) in cell suspension (B) were identified by CD45/PerCp+. The subpopulation of cell suspension in APC/FITC dotplot (C), macrophages (red), PMNs (blue) and lymphocytes (green) are identified by CD14/APC+ gate in APC/SSC plot (D), CH138A/FITC+ gate in FITC/SSC plot (E), and FSC/SSC size/granularity gate (F), respectively.
Fig 3.
Flow cytometry identification of differential somatic cell count and simultaneous their quantification of all cells and each cell type.
(A)(B)(C) fresh blood cell suspension, (D)(E)(F) milk cell suspension after 80°C × 30 min and (G)(H)(I)milk cell suspension conserved at 4°C in PBS pH 7.4. The cell subpopulations of each sample were shown in APC/FITC dotplot (A)(D)(G), the viable and non-viable population of all cells and each subpopulation were shown in histogram-Vioblue scatter (B)(E)(H) and (C)(F)(I), respectively.
Fig 4.
Cell viability of reference cell suspension (4°C) and of identical cell suspension with different heat treatments (39°C, 60°C, 80°C during 30 min).
(A) Boxplot and whiskers of flow cytometry results with milk somatic cell suspensions incubated with vioblue Live/Dead kit (n = 4). (B) Cell viability of each type cell for 4°C (white bars), 39°C (hatched bars), 60°C (grey bars), 80°C (dark bars) centrifugations by flow cytometry; (C) Microscopy images (objective ×10) of milk somatic cell suspensions incubated with syto 9 and propidium iodide Live/Dead kit (n = 9). Means with different superscripts (a and b) differ significantly (P<0.001).
Fig 5.
Cell viability with various centrifugation rates (400, 1500, 3000, 5000×g during 10 min at 4°C) comparing to the reference milk cell suspension without supplementary centrifugation (Ref).
(A) Boxplot and whiskers of flow cytometry results with milk somatic cell suspensions incubated with specific antibodies and vioblue live/dead kit (n = 4); (B) Cell viability of each type cell for 400×g (white bars), 1500×g (hatched bars), 3000×g (grey bars), 5000×g (dark bars) centrifugations by flow cytometry; (C) Mean proportion of macrophages (dark sectors), PMNs (grey sectors) and lymphocytes (white sectors) in cell samples under various centrifugation with different gravitational velocities. Means with different superscripts (a-d) differ significantly (P<0.05).
Table 2.
Flow cytometry results concerning the proportion of cell subpopulation and their viabilities of milk cell suspensions in PBS and milk conservation during 24, 48, 72h (n = 4).