Clia Biochern, Vol. 25, pp. 277-283, 1992 Printed in the USA. All rights reserved.
0009-9120/92 $5.00 + .00 Convri~ht © 1992 The Canadian Society of Clinical Chemists.
Flow Cytometric Measurement of Intracellular Bilirubin in Human Peripheral Blood Mononuclear Cells Exposed to Unconjugated Bilirubin YOSHIO HAGA, H. DAVID KAY, MARGARET A. TEMPERO, and ROWEN K. ZETTERMAN Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198 and the Omaha Veterans Administration Medical Center, Omaha, NE 68105, USA Human peripheral blood mononuclear cells were incubated at 37 °C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p < 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both -1::3
/ o z
/
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0
,_o.o_O-O ........ 0 50 I0O 150 200 CONCENTRATION OF 81LIRUBIN USED TO TREAT CELLS (/~rnol/L)
BY F A C S c A N
After treatment of PBMNC with bilirubin, the cells were analyzed for a u t o f l u o r e s c e n c e using FACScan. When FACScan was aligned using "Autocomp" software with calibrate beads, a significant autofluorescence was detected in both FL1 (530 nm) and FL2 (585 nm) channels, and the intensity of the FL2 autofluorescence was usually higher than that of the FL1 autofluorescence (Figures 4A and 5A). The intensity of autofluorescence in both FL1 and FL2 channels increases or decreases when the photomultiplier tube voltage is turned up or down, respectively. The autofluorescence can be adjusted by fluorescence compensation using "Consort 30" software so as to be detected only in the FL2 channel without significant change of intensity of the FL2 signal (Figures 4B and 5B). The values of FL2 auto3.0
2.0
F i g u r e 3 - - Change of q u a n t i t y of i n t r a c e l l u l a r b i l i r u b i n incorporated into b i l i r u b i n - t r e a t e d PBMNC. P B M N C (10 ? cells} t r e a t e d at 37 °C for 60 min at 5 x 10 s cells/mL with various concentrations of b i l i r u b i n in 30 g/L BSA were homogenized in 1.0 mL of c h l o r o f o r m - m e t h a n o l (2:1, v/v) solution. The q u a n t i t y of i n t r a c e l l u l a r b i l i r u b i n in each extract was d e t e r m i n e d from the i n t e n s i t y of absorbance at 450 nm.
fluorescence presented in the rest of this paper were obtained after this compensation. The amount of autofluorescence detected in the FL2 channel (at 585 nm) started to increase at 51 ~mol/L, but the increase tended to plateau from 137 to 205 ~mol/L (Figure 5). S u b s e q u e n t l y , FL2 a u t o f l u o r e s c e n c e and t h e quantity of intracellular bilirubin measured by the chloroform-methanol extraction method were concomitantly assayed in various samples of bilirubintreated PBMNC. As shown in Figure 6, a significant correlation was observed between their levels, suggesting that the amount of intracellular bilirubin
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ANALYSIS OF B I L I R U B I N - T R E A T E D PBMNC
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0009-9120/92 $5.00 + .00 Convri~ht © 1992 The Canadian Society of Clinical Chemists.
Flow Cytometric Measurement of Intracellular Bilirubin in Human Peripheral Blood Mononuclear Cells Exposed to Unconjugated Bilirubin YOSHIO HAGA, H. DAVID KAY, MARGARET A. TEMPERO, and ROWEN K. ZETTERMAN Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198 and the Omaha Veterans Administration Medical Center, Omaha, NE 68105, USA Human peripheral blood mononuclear cells were incubated at 37 °C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p < 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both -1::3
/ o z
/
/ ©
0
,_o.o_O-O ........ 0 50 I0O 150 200 CONCENTRATION OF 81LIRUBIN USED TO TREAT CELLS (/~rnol/L)
BY F A C S c A N
After treatment of PBMNC with bilirubin, the cells were analyzed for a u t o f l u o r e s c e n c e using FACScan. When FACScan was aligned using "Autocomp" software with calibrate beads, a significant autofluorescence was detected in both FL1 (530 nm) and FL2 (585 nm) channels, and the intensity of the FL2 autofluorescence was usually higher than that of the FL1 autofluorescence (Figures 4A and 5A). The intensity of autofluorescence in both FL1 and FL2 channels increases or decreases when the photomultiplier tube voltage is turned up or down, respectively. The autofluorescence can be adjusted by fluorescence compensation using "Consort 30" software so as to be detected only in the FL2 channel without significant change of intensity of the FL2 signal (Figures 4B and 5B). The values of FL2 auto3.0
2.0
F i g u r e 3 - - Change of q u a n t i t y of i n t r a c e l l u l a r b i l i r u b i n incorporated into b i l i r u b i n - t r e a t e d PBMNC. P B M N C (10 ? cells} t r e a t e d at 37 °C for 60 min at 5 x 10 s cells/mL with various concentrations of b i l i r u b i n in 30 g/L BSA were homogenized in 1.0 mL of c h l o r o f o r m - m e t h a n o l (2:1, v/v) solution. The q u a n t i t y of i n t r a c e l l u l a r b i l i r u b i n in each extract was d e t e r m i n e d from the i n t e n s i t y of absorbance at 450 nm.
fluorescence presented in the rest of this paper were obtained after this compensation. The amount of autofluorescence detected in the FL2 channel (at 585 nm) started to increase at 51 ~mol/L, but the increase tended to plateau from 137 to 205 ~mol/L (Figure 5). S u b s e q u e n t l y , FL2 a u t o f l u o r e s c e n c e and t h e quantity of intracellular bilirubin measured by the chloroform-methanol extraction method were concomitantly assayed in various samples of bilirubintreated PBMNC. As shown in Figure 6, a significant correlation was observed between their levels, suggesting that the amount of intracellular bilirubin
A
z
< m nO m ms 1.0
0
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~--- t . J
ANALYSIS OF B I L I R U B I N - T R E A T E D PBMNC
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