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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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Opsonophagocytic-killing assay activity of HL-60 cells during 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 applied to a conventional pneumococcal OPKA against serotype 19F as described in the Materials and Methods. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
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Figure 3: Opsonophagocytic-killing assay activity of HL-60 cells during 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 applied to a conventional pneumococcal OPKA against serotype 19F as described in the Materials and Methods. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.

Mentions: To further investigate the correlation between phenotypic change of the cells and OPKA activity during differentiation, a standard OPKA was performed using serotype 19F of S. pneumoniae. Undifferentiated HL-60 cells showed 49.7% of OPKA activity as determined by percent killing. With DMF, OPKA activity increased significantly on day 1 and remained constant till day 5. On the contrary, ATRA and VitD3 gradually increased the OPKA activity as the differentiation continued. At the end of differentiation, there was no significant difference in OPKA activity among the 3 agents and the titer reached approximately 90% (Fig. 3). Pearson correlation analysis demonstrated that changes in CD11c expression of the differentiated HL-60 cells correlated well with OPKA activity in all 3 agents (Table 2). With DMF, CD32 expression had a strong correlation with the OPKA activity and the expression of other markers such as CD14, CD11c, and CD18 demonstrated a moderate correlation with the OPKA activity compared with other agents. CD14 showed a strong correlation with the OPKA activity on the differentiation of HL-60 cells induced by ATRA treatment.


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)

Opsonophagocytic-killing assay activity of HL-60 cells during 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 applied to a conventional pneumococcal OPKA against serotype 19F as described in the Materials and Methods. 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 3: Opsonophagocytic-killing assay activity of HL-60 cells during 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 applied to a conventional pneumococcal OPKA against serotype 19F as described in the Materials and Methods. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
Mentions: To further investigate the correlation between phenotypic change of the cells and OPKA activity during differentiation, a standard OPKA was performed using serotype 19F of S. pneumoniae. Undifferentiated HL-60 cells showed 49.7% of OPKA activity as determined by percent killing. With DMF, OPKA activity increased significantly on day 1 and remained constant till day 5. On the contrary, ATRA and VitD3 gradually increased the OPKA activity as the differentiation continued. At the end of differentiation, there was no significant difference in OPKA activity among the 3 agents and the titer reached approximately 90% (Fig. 3). Pearson correlation analysis demonstrated that changes in CD11c expression of the differentiated HL-60 cells correlated well with OPKA activity in all 3 agents (Table 2). With DMF, CD32 expression had a strong correlation with the OPKA activity and the expression of other markers such as CD14, CD11c, and CD18 demonstrated a moderate correlation with the OPKA activity compared with other agents. CD14 showed a strong correlation with the OPKA activity on the differentiation of HL-60 cells induced by ATRA treatment.

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