Labeling:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) ..
Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
Saline:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) ..
Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
Avidin-Biotin Assay:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) ..
Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
Staining:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) ..
Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
Flow Cytometry:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) ..
Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
Expressing:Article Title: Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering
Article Snippet: .. Materials Jurkat clone E6-1 (ATCC, cat. no. TIB-152; see Support Protocol 1 for culture and harvesting) Complete RPMI culture medium (see recipe) 10 mM Ac 5 ManNTGc ( 1 ) stock solution for thiol labeling experiments and/or Ac 4 ManNLev ( Kim et al., 2004 ) [or 1,3,4- O -Bu 3 ManNLev ( Aich et al., 2008 )) stock solution for ketone labeling] Ethanol (EtOH; 200 proof) Phosphate-buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010-049) Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706) MB solution (see recipe), freshly prepared Biotin buffer (see recipe), freshly prepared 5.0 mM biotin hydrazide stock solution (see recipe) Avidin staining buffer (ASB; see recipe) Fluorescein isothiocyanate (FITC)-labeled avidin stock solution (see recipe) Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells 6-well tissue culture plates Centrifuge Tubes for flow cytometer (5-ml polystyrene round-bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054) Flow cytometer equipped with a 488-nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences) Incubate cells with sugar analogs 1 Incubate Jurkat cells with sugar analog (e.g., 1 for thiol expression or Ac 4 ManNLev or1,3,4- O -Bu 3 ManNLev ) as described in Basic Protocol 1, but use 6-well tissue-culture plates (or 35-mm dishes) instead of 24-well plates, and seed ~1.0 × 10 6 cells in 2.0 ml for each sample to be analyzed. ..
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