Technical Reference #1848

1848.

A Threonine-Based Targeting Signal in the Human CD1d Cytoplasmic Tail Controls Its Functional Expression Jianyun Liu; Daniel Shaji; Sungyoo Cho; Wenjun Du; Jacquelyn Gervay-Hague and Randy R. Brutkiewicz, Indiana University School of Medicine, Journal of Immunology, 184(1848), (2010)
Link To Paper

Materials & Methods:
Staining for confocal microscopy was performed as described previously (15). Briefly HEK293 cells were plated in sterile glass-bottom 35-mm dishes coated with collagen (MatTek Ashland MA). After the cells became 50–80% confluent they were washed fixed and then permeabilized by 0.1% saponin in HBSS with 0.1% BSA (HBSS/BSA). To stain the CD1d–b2m complex cells were incubated with the CD1d–b2m complexspecific 42.1 mAb followed by a Texas Red-conjugated donkey anti-mouse Ig antiserum. After the free reactive sites were blocked with normal mouse serum (Sigma-Aldrich St. Louis MO) the cells were incubated with a FITC-conjugated anti-human LAMP-1 mAb. To stain the free CD1d HC the cells were incubated with the anti-CD1d freeHCmAbC3D5 followed by a FITC-conjugated anti-mouse Ig antiserum. ER-specific staining was performed by incubating the cells with a rabbit anti-Sec61b polyclonal antiserum followed by a Texas Red-conjugated anti-rabbit Ig antiserum. For the nucleus staining cells were immersed in HBSS/BSA with 0.1% saponin containing Hoechst (1:2000) for 5 min. Just prior to confocal analysis the cells were placed in mounting medium (10 mM Tris [pH 8.5] and 2% 14- diazabicyclo[2.2.2]octane). The cells were viewed on a LSM-510 laser scanning confocal microscope (Zeiss Thornwood NY) modified for onephoton microscopy using an oil immersion lens at 3100. Analysis of the relative level of CD1d colocalization with LAMP-1 was performed using MetaMorph software (version 5; Molecular Devices Sunnyvale CA).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HEK293 cells