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. 2000 Jun 5;191(11):1895–1904. doi: 10.1084/jem.191.11.1895

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Long life span of CD1d1–glycolipid complexes. (A) Plate-bound CD1d1 (2.5 μg/ml) was loaded with 0.1 or 0.3 μM αGalCer for 2 h, washed, and chased for different periods of time before adding the Vα14-Jα281 NKT cell hybridoma DN32D3 and measuring IL-2 release in the supernatant (mean ± SD). (B) Plate-bound CD1d1 was first incubated with 30 μg/ml of LAM or GM1 for 6 h, washed, and chased for different periods of time. 0.1 μM αGalCer was added for 2 h and washed before incubation with DN32D3. (C) Microwells were coated overnight with 2.5 μg/ml CD1d1 or BSA or 0.1μM αGalCer, washed, then pulsed for 2 h with 0.1 μM αGalCer as indicated, and washed again before adding the CD1d-restricted Vα14 hybridomas DN32D3 or a control CD1d-restricted non-Vα14 hybridoma, 431.A11 (Vα3.2-Jα8/Vβ8), and measuring IL-2 release (mean ± SD). 431A11 and DN32D3 produced similar amounts of IL-2 when stimulated with CD1d1-expressing thymocytes.

Long life span of CD1d1–glycolipid complexes. (A) Plate-bound CD1d1 (2.5 μg/ml) was loaded with 0.1 or 0.3 μM αGalCer for 2 h, washed, and chased for different periods of time before adding the Vα14-Jα281 NKT cell hybridoma DN32D3 and measuring IL-2 release in the supernatant (mean ± SD). (B) Plate-bound CD1d1 was first incubated with 30 μg/ml of LAM or GM1 for 6 h, washed, and chased for different periods of time. 0.1 μM αGalCer was added for 2 h and washed before incubation with DN32D3. (C) Microwells were coated overnight with 2.5 μg/ml CD1d1 or BSA or 0.1μM αGalCer, washed, then pulsed for 2 h with 0.1 μM αGalCer as indicated, and washed again before adding the CD1d-restricted Vα14 hybridomas DN32D3 or a control CD1d-restricted non-Vα14 hybridoma, 431.A11 (Vα3.2-Jα8/Vβ8), and measuring IL-2 release (mean ± SD). 431A11 and DN32D3 produced similar amounts of IL-2 when stimulated with CD1d1-expressing thymocytes.