Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.
Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.
Yen
C F
CF
Regional Research Laboratory, Kaiser Foundation Hospitals, Los Angeles CA 90027.
Kalunta
C I
CI
Chen
F S
FS
Kaptein
J S
JS
Lin
C K
CK
Lad
P M
PM
D001402:B-Lymphocytes / Q000737:chemistry*; D001692:Biological Transport; D002232:Carbocyanines; D002460:Cell Line; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D011973:Receptors, LDL / Q000032:analysis*; D013601:T-Lymphocytes / Q000737:chemistry*; D014168:Transferrin / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D014168:Transferrin; C024286:3,3'-dioctadecylindocarbocyanine