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Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.
Cytometry A. 2005 Jun; 65(2):103-15.C

Abstract

BACKGROUND

Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing.

METHODS

Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets.

RESULTS

In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets.

CONCLUSION

With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

Authors+Show Affiliations

Interdisciplinary Center for Clinical Research Leipzig, University of Leipzig, Leipzig, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15830380

Citation

Mittag, Anja, et al. "Polychromatic (eight-color) Slide-based Cytometry for the Phenotyping of Leukocyte, NK, and NKT Subsets." Cytometry. Part a : the Journal of the International Society for Analytical Cytology, vol. 65, no. 2, 2005, pp. 103-15.
Mittag A, Lenz D, Gerstner AO, et al. Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets. Cytometry A. 2005;65(2):103-15.
Mittag, A., Lenz, D., Gerstner, A. O., Sack, U., Steinbrecher, M., Koksch, M., Raffael, A., Bocsi, J., & Tárnok, A. (2005). Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets. Cytometry. Part a : the Journal of the International Society for Analytical Cytology, 65(2), 103-15.
Mittag A, et al. Polychromatic (eight-color) Slide-based Cytometry for the Phenotyping of Leukocyte, NK, and NKT Subsets. Cytometry A. 2005;65(2):103-15. PubMed PMID: 15830380.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets. AU - Mittag,Anja, AU - Lenz,Dominik, AU - Gerstner,Andreas O H, AU - Sack,Ulrich, AU - Steinbrecher,Michael, AU - Koksch,Mario, AU - Raffael,Alexander, AU - Bocsi,Jozsef, AU - Tárnok,Attila, PY - 2005/4/15/pubmed PY - 2005/9/15/medline PY - 2005/4/15/entrez SP - 103 EP - 15 JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology JO - Cytometry A VL - 65 IS - 2 N2 - BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time. SN - 1552-4922 UR - https://www.unboundmedicine.com/medline/citation/15830380/Polychromatic__eight_color__slide_based_cytometry_for_the_phenotyping_of_leukocyte_NK_and_NKT_subsets_ L2 - https://doi.org/10.1002/cyto.a.20140 DB - PRIME DP - Unbound Medicine ER -