Purified EVs were concentrated by ultracentrifugation (18 h at 100,000 × g) and particles (5 × 108/mL incubated with 2 μM PKH26 Red Fluorescent cell dye (MINI26, Sigma-Aldrich). After 30 min at 37°C (dark), the mixture was diluted 5x with PBS and ultracentrifuged (18 h, 100,000 × g). The pellet was resuspended with Diluent C, and the number of EVs determined by NTA. Different concentrations of labeled EVs were incubated with trypomastigotes (1 × 106/mL) in DMEM containing 5% glucose. Parasites were collected by centrifugation at different time points (5 min at 1,000 × g), washed once with PBS, and resuspended in PBS-0.5% p-formaldehyde. Samples were analyzed by flow cytometry (Fortessa, BD).
Pkh26 red fluorescent cell dye
Manufactured by Merck Group
Sourced in Germany
PKH26 Red Fluorescent cell dye is a lipophilic fluorescent dye that can be used to label the cell membrane of living cells. It has an excitation maximum at 551 nm and an emission maximum at 567 nm, producing a red fluorescent signal.
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Lab products found in correlation
2 protocols using pkh26 red fluorescent cell dye
1
Labeling and Uptake of Extracellular Vesicles by Trypomastigotes
2
Murine and Human Leukemia Phagocytosis Assays
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For mouse phagocytosis assays, c-Kit+ dsRed+ murine MLL–AF9 leukemia cells were added to macrophage cultures in a 2:1 ratio. After 18 h, the cells were stained with a BV421–F4/80 antibody (BioLegend, San Diego, CA, USA), and the percentage of F4/80+dsRed+ cells was determined by FACS analysis.
For the CD47 blocking experiments, we incubated c-Kit+ dsRed+ murine MLL–AF9 leukemia cells for 30 min with an anti-CD47 antibody or rat IgG2a isotype control (30 mg/mL; BioXCell, Lebanon, NH, USA), before co-culture with macrophages for 1 h at 37°C. The percentage of F4/80+dsRed+ cells was determined by flow cytometry as described above.
For human phagocytosis assays, we labeled human leukemia cell lines with the PKH67 green fluorescent cell dye according to the manufacturer’s instructions (Sigma-Aldrich, Darmstadt, Germany) and stained macrophages with the PKH26 red fluorescent cell dye (Sigma-Aldrich). AML cells were mixed with human macrophages in a 2:1 ratio and incubated for either 2 h (Mono Mac 6 cells) or 18 h (MA9:16 cells). The percentage of PKH26+ PKH67+ macrophages was determined by FACS.
For the CD47 blocking experiments, we incubated c-Kit+ dsRed+ murine MLL–AF9 leukemia cells for 30 min with an anti-CD47 antibody or rat IgG2a isotype control (30 mg/mL; BioXCell, Lebanon, NH, USA), before co-culture with macrophages for 1 h at 37°C. The percentage of F4/80+dsRed+ cells was determined by flow cytometry as described above.
For human phagocytosis assays, we labeled human leukemia cell lines with the PKH67 green fluorescent cell dye according to the manufacturer’s instructions (Sigma-Aldrich, Darmstadt, Germany) and stained macrophages with the PKH26 red fluorescent cell dye (Sigma-Aldrich). AML cells were mixed with human macrophages in a 2:1 ratio and incubated for either 2 h (Mono Mac 6 cells) or 18 h (MA9:16 cells). The percentage of PKH26+ PKH67+ macrophages was determined by FACS.
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