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Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae.

Kim KH, Seoh JY, Cho SJ - J. Korean Med. Sci. (2015)

Bottom Line: Differentiation with ATRA or VitD3 increased the respiratory burst after day 4.DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased.Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, School of Medicine, Ewha Womans University, Seoul, Korea.

ABSTRACT
Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

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Related in: MedlinePlus

Analysis of respiratory burst during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD3 (20 µg/mL) for the indicated time period. The HL-60 cells were incubated with 50 mM luminal and 500 nM PMA for 2 hr. Chemiluminescence intensity was examined with a luminometer. One of the three similar results is shown. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
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Figure 2: Analysis of respiratory burst during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD3 (20 µg/mL) for the indicated time period. The HL-60 cells were incubated with 50 mM luminal and 500 nM PMA for 2 hr. Chemiluminescence intensity was examined with a luminometer. One of the three similar results is shown. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.

Mentions: During phagocytosis, a metabolic process known as the respiratory burst occurs in the phagocytes producing a number of reactive oxygen species that are extremely toxic to the microorganisms. Respiratory burst of the differentiated HL-60 cells was determined by the chemiluminescence assay. The production of reactive oxygen species was immediately down-regulated at the early stage of differentiation (up to 2 days after induction). Then, DMF showed a robust induction of the respiratory burst on day 3 and the induction reached its maximum on day 4. ATRA and VitD3 showed similar patterns with DMF in the kinetics of the respiratory burst but produced 10 times higher intensities of the fluorescence on day 4 and 5 compared to DMF (Fig. 2).


Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae.

Kim KH, Seoh JY, Cho SJ - J. Korean Med. Sci. (2015)

Analysis of respiratory burst during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD3 (20 µg/mL) for the indicated time period. The HL-60 cells were incubated with 50 mM luminal and 500 nM PMA for 2 hr. Chemiluminescence intensity was examined with a luminometer. One of the three similar results is shown. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4310939&req=5

Figure 2: Analysis of respiratory burst during HL-60 differentiation. HL-60 cells were incubated with or without DMF (0.8%), ATRA (1 µM), or VitD3 (20 µg/mL) for the indicated time period. The HL-60 cells were incubated with 50 mM luminal and 500 nM PMA for 2 hr. Chemiluminescence intensity was examined with a luminometer. One of the three similar results is shown. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
Mentions: During phagocytosis, a metabolic process known as the respiratory burst occurs in the phagocytes producing a number of reactive oxygen species that are extremely toxic to the microorganisms. Respiratory burst of the differentiated HL-60 cells was determined by the chemiluminescence assay. The production of reactive oxygen species was immediately down-regulated at the early stage of differentiation (up to 2 days after induction). Then, DMF showed a robust induction of the respiratory burst on day 3 and the induction reached its maximum on day 4. ATRA and VitD3 showed similar patterns with DMF in the kinetics of the respiratory burst but produced 10 times higher intensities of the fluorescence on day 4 and 5 compared to DMF (Fig. 2).

Bottom Line: Differentiation with ATRA or VitD3 increased the respiratory burst after day 4.DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased.Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, School of Medicine, Ewha Womans University, Seoul, Korea.

ABSTRACT
Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Show MeSH
Related in: MedlinePlus