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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 apoptotic cell death 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. Cells were stained with 20 µg/mL of 7-AAD and percentage of apoptotic cell populations were determined by analyzing fluorescence intensity of moderately stained cells with flow cytometry. *Indicates P < 0.05 when the values were compared with that of the day 0. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
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Figure 1: Analysis of apoptotic cell death 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. Cells were stained with 20 µg/mL of 7-AAD and percentage of apoptotic cell populations were determined by analyzing fluorescence intensity of moderately stained cells with flow cytometry. *Indicates P < 0.05 when the values were compared with that of the day 0. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.

Mentions: Cells stop proliferation during differentiation and begin to die at a certain time point after the end of differentiation. In order to examine the change in dead cell populations during differentiation, apoptotic cells were enumerated using 7-AAD, a fluorescent dye intercalating into DNA of dead cell. Dead cell fraction of HL-60 cells before the differentiation was 1.8% and it remained less than 10% for 3 days when the cells were differentiated with DMF or ATRA. However, apoptotic cells increased on day 4 and 5 post DMF or ATRA treatment. On the contrary to DMF and ATRA, VitD3 showed no increase of dead cells until day 4 with a small increase in the dead cells (3.5%) on day 5 (Fig. 1).


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 apoptotic cell death 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. Cells were stained with 20 µg/mL of 7-AAD and percentage of apoptotic cell populations were determined by analyzing fluorescence intensity of moderately stained cells with flow cytometry. *Indicates P < 0.05 when the values were compared with that of the day 0. 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 1: Analysis of apoptotic cell death 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. Cells were stained with 20 µg/mL of 7-AAD and percentage of apoptotic cell populations were determined by analyzing fluorescence intensity of moderately stained cells with flow cytometry. *Indicates P < 0.05 when the values were compared with that of the day 0. DMF, dimethylformamide; ATRA, all-trans retinoic acid; VitD3, vitamin D3; 7-ADD, 7-aminoactinomycin D.
Mentions: Cells stop proliferation during differentiation and begin to die at a certain time point after the end of differentiation. In order to examine the change in dead cell populations during differentiation, apoptotic cells were enumerated using 7-AAD, a fluorescent dye intercalating into DNA of dead cell. Dead cell fraction of HL-60 cells before the differentiation was 1.8% and it remained less than 10% for 3 days when the cells were differentiated with DMF or ATRA. However, apoptotic cells increased on day 4 and 5 post DMF or ATRA treatment. On the contrary to DMF and ATRA, VitD3 showed no increase of dead cells until day 4 with a small increase in the dead cells (3.5%) on day 5 (Fig. 1).

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