Multiparameter flow cytometry of a pH sensitive ligand bound to receptors and inside cells
- Univ. of New Mexico, Albuquerque (United States) Los Alamos National Lab., NM (United States)
Because fluoresceinated ligands of the neutrophil formyl peptide receptor can be protonated either upon binding to the receptor on the cell surface or in acidified intracellular compartments, the authors synthesized a ligand conjugated to the pH sensitive fluorescent probe SNAFL (CHO-Met-Leu-Phe-Phe-Lys-SNAFL). In the three laser flow cytometer at LANL, protonated dye is excited at 488 nm and emits at 530 nm; unprotonated dye is excited at 568 nm and emits at 650 nm. Detection at the isobestic and isoemissive points at 528 and 600 nm is used to keep track of variations in ligand concentration from sample to sample. The SNAFL-ligand bound to HL-60 cells (which overexpress the formyl peptide receptor) was compared to the free ligand in solution over a pH range from 6.5 to 9.0. The results suggest that the ligand bound to cell surface receptors was protonated in the binding pocket, possibly by virtue of its proximity to His 90, based on sequence data. When the cells were raised from 4[degrees] to 37[degrees], they also observed a time-dependent acidification of the ligand, indicative of ligand-receptor processing beginning 3-4 minutes after internalization.
- OSTI ID:
- 6831855
- Report Number(s):
- CONF-9303114-; CODEN: CYTODQ; CNN: AI19032; RR 01315
- Journal Information:
- Cytometry (Baltimore); (United States), Vol. 6; Conference: 16. congress of the International Society for Analytical Cytology, Colorado Springs, CO (United States), 21-26 Mar 1993; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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LIGANDS
ACIDIFICATION
CHEMICAL BONDS
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FLUORESCENCE
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NEUTROPHILS
PH VALUE
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BLOOD
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LEUKOCYTES
LUMINESCENCE
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MEMBRANE PROTEINS
NATIONAL ORGANIZATIONS
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US DOE
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550200* - Biochemistry