Table 1.
The characteristic of liver tissue used and the main results of liver cells isolations.
Table 2.
Antibodies used in this study.
Fig 1.
Populations of cells in liver isolates (light scatters).
Exemplary flow cytometric dot plots showing populations of cells isolated from human liver tissue in light scatters: forward—FSC and side—SSC. Depending on specimen, there are minimum 2 and maximum 4 distinct cells populations (labeled P2 –P5) identified in the P1 gate (cellular debris and doublets excluded). Population P2 –blue dots; P3 –yellow dots; P4 –green dots; P5 –purple dots. The percentage of cells in the individual populations of the P1 gate—specimen H27-14: P2 = 35.9%, P3 = 62.8%; H26-14: P2 = 32.3%, P3 = 52.8%, P4 = 6.1%; H16-13: P2 = 47.6%, P3 = 46.4%, P4 = 1.7%, P5 = 1.3%; H22-13: P2 = 40.6%, P3 = 41.6%, P4 = 11%, P5 = 1.1%.
Fig 2.
Staining of the isolated cells with fluorophore-conjugated specific antibodies.
Exemplary immunostainings of the cells isolated from liver specimen (H22-13, see Table 1). (A) The cells labeled with anti-Alb (left panel) and anti-A1AT (right panel) antibodies were found predominantly in the P2 population (97.5% and 86.2% of the P2, respectively) and identified as hepatocytes. (B) Nonparenchymal liver cells were stained with anti-CD45 (populations P3 = 80.3% and P4 = 74.1%, left panel) and anti-CD14 antibodies (population P4 = 43.4%, right panel) (see Table 2 for the list of antibodies). Alb- and A1AT-positive cells–green dots; CD45-positive cells–pink dots; CD14-positive cells–blue dots. Abbr.: Alb, albumin; A1AT, α-1-antitrypsin; FSC, forward scatter; SSC, side scatter.
Fig 3.
Double immunostaining of the isolated cells.
Exemplary double immunostainings of the cells isolated from liver specimen (H31-14, see Table 1). (A) In the P2 population cells labeled with anti-Alb antibody expressed neither CD45 (83.5% vs.1.3%, left panel) nor CD14 (81.3% vs. 0.5%, right panel) markers. (B) Nonparenchymal cells in the P3 population (left panel) expressed CD45 marker but not CD14 (46.7% vs. 0.1%). Some of the cells from the P4 population (right panel) expressed both CD45 and CD14 markers (CD45 = 23.9%, CD14 = 16%, and CD45/CD14 = 15.1%) (see Table 2 for the list of antibodies). Alb-positive cells–green dots; CD45-positive cells–pink dots; CD14-positive cells–blue dots; CD54/CD14-positive cells–grey dots; unstained cells–yellow dots. Abbr.: Alb, albumin; FITC, PerCP, APC—fluorophores.
Fig 4.
The impact of sex, chemotherapy, and age on the percentage of Alb-positive cells in P2 population.
Comparison of the percentage of Alb-positive cells in the P2 population between groups of patients. (A) F (n = 17), M (n = 18); Ch- (n = 9), CH+ (n = 26); A60- (n = 18), A60+ (n = 17). (B) F Ch+ (n = 13), M Ch+ (n = 13), F Ch- (n = 4), M Ch- (n = 5). (C) F A60- (n = 11), F A60+ (n = 6), M A60- (n = 7), M A60+ (n = 11). The differences between two independent groups were analyzed using the Welch's two-sample t-test. For multiple comparisons the Benjamini & Hochberg correction procedure was applied. In multivariate analysis the two-way Analysis of Variance (ANOVA) for linear model with interaction term was used. ANOVA results were subsequently confirmed using the Tukey HSD test. The results are presented as the median values with the range for each variable. * p < 0.05 Abbr.: Alb, albumin; F, female; M, male; Ch-, non-treated patients; Ch+, patients after chemotherapy; A60-, patients under 60 years old; A60+, patients 60 years old and older.
Table 3.
The p-values for selected interactions from two-way Analysis of Variance (ANOVA) confirmed using Tukey HSD test.
Fig 5.
The impact of sex, chemotherapy, and age on the percentage of A1AT-positive cells in P2 population.
Comparison of the percentage of A1AT-positive cells in the P2 population between groups of patients. F (n = 15), M (n = 14), Ch- (n = 7), Ch+ (n = 22), A60- (n = 16), A60+ (n = 13). The differences between two independent groups were analyzed using the Welch's two-sample t-test. The results are presented as the median values with the range for each variable. Abbr.: A1AT, α-1-antitrypsin; F, female; M, male; Ch-, non-treated patients; Ch+, patients after chemotherapy; A60-, patients under 60 years old; A60+, patients 60 years old and older.