classical monocyte isolation kit  (Miltenyi Biotec)

Journal: HemaSphere

Article Title: Monocyte‐mediated metabolic rewiring via CD31‐CD38 interactions promotes growth and drug‐resistance in multiple myeloma

doi: 10.1002/hem3.70358

Figure Lengend Snippet: Monocytes transfer mitochondria via extracellular vesicles . (A) Western blot analysis of EV markers CD63 and CD81 in THP‐1 EVs and THP‐1 cell lysate. CD81 and CD63 were detected in the EV fraction, confirming successful EV isolation. (B) Quantification of CD31 protein in THP‐1‐derived EVs ( n = 4) by ELISA. EVs derived from HEK293T cells ( n = 3) were used as negative controls. (C) Solvent control or daratumumab pretreated (Dara; α‐CD38) BM‐MNCs obtained from four newly diagnosed MM (NDMM) patients were incubated with MTDR + Mt‐EVs isolated from PBMC‐derived monocyte culture supernatant (Mt‐EV), or anti‐CD31 pretreated MTDR + Mt‐EVs (α‐CD31), or sonicated MTDR + Mt‐EVs (sonicated Mt‐EV). After 12 h, the MTDR signal in MM cells (CD138 + CD38 + CD33 − ) was measured by flow cytometry. Data are presented as mean ± SD. Each data point represents an individual patient sample . P‐values were calculated using one‐way ANOVA with Tukey's post hoc test. (D–G) Solvent control or daratumumab pretreated (Dara; α‐CD38) MM cell lines, (D) UM9, (E) RPMI‐8226, (F) MM.1S, and (G) U266, were incubated with MTDR + Mt‐EVs isolated from THP‐1 monocyte culture supernatant (Mt‐EV), or anti‐CD31 pretreated MTDR + Mt‐EVs (α‐CD31), or sonicated MTDR + Mt‐EVs (sonicated Mt‐EV). After 12 h, the MTDR signal in MM cell lines was measured by flow cytometry. Data are presented as mean ± SD of four independent experiments, performed in duplicate. P‐values were calculated using one‐way ANOVA with Tukey's post hoc test. (H, I) Solvent control or daratumumab pretreated (Dara; α‐CD38) UM9 cells were incubated with unlabeled Mt‐EVs isolated from THP‐1 monocyte culture supernatant (Mt‐EV), or anti‐CD31 pretreated MTDR + Mt‐EVs (α‐CD31), or with sonicated Mt‐EVs (sonicated Mt‐EV). After 12 h, the absolute mitochondrial membrane potential (H) was assessed using the MTDR probe, and the total mitochondrial content (I) of UM9 cells was measured using the MTG probe with flow cytometry. Data are presented as mean ± SD of four independent experiments, each performed in duplicate or triplicate. P‐values were calculated using one‐way ANOVA with Tukey's post hoc test. (J) Representative dot plots illustrate the gating strategy used to identify CD138 + MM cells in BM‐MNCs from patients co‐cultured with MTDR + Mt‐EVs. CD138 + MM cells were gated based on morphology (FSC‐A vs. SSC‐A), followed by selection of single cells (FSC‐A vs. FSC‐H) and live cells (Near‐IR negative cells). Transfer of MTDR + Mt‐EVs to MM cells was assessed by measuring MTDR fluorescence in all CD138 + MM cells after 12 h of co‐culture. Representative flow cytometry histogram overlays show MTDR signal in all CD138 + MM under different treatment conditions: Mt‐EV, Mt‐EV + Dara, Mt‐EV + α‐CD31, and Sonicated Mt‐EV (top to bottom). (K) Representative dot plots show the gating strategy used to identify MM cell lines co‐cultured with MTDR + Mt‐EVs. Cells were gated based on morphology (FSC‐A vs. SSC‐A), followed by selection of single cells (FSC‐A vs. FSC‐H) and live cells (Near‐IR negative cells). Subsequently, CD33⁻CD138 + MM cells were distinguished. Transfer of MTDR + Mt‐EVs to MM cells was assessed by measuring the MTDR fluorescence in MM cells after 12 h of co‐culture. Representative flow cytometry histogram overlays show MTDR signal in MM under different treatment conditions: Mt‐EV, Mt‐EV + Dara, Mt‐EV + α‐CD31, and Sonicated Mt‐EV (top to bottom). A P < 0.05 was considered statistically significant, and each significance is indicated with *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. ANOVA, analysis of variance; BM‐MNCs, bone marrow mononuclear cells; ELISA, enzyme‐linked immunosorbent assay; EV, extracellular vesicles; MM, multiple myeloma; MTDR, MitoTracker Deep Red; PBMC, peripheral blood mononuclear cells.

Article Snippet: PBMCs were enriched for monocytes using immunomagnetic MACS magnetic beads coated with antibodies against CD14, according to the manufacturer's instructions (pan Monocyte Isolation Kit; Miltenyi; Cat# 130‐117‐337).

Techniques: Western Blot, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Solvent, Control, Incubation, Sonication, Flow Cytometry, Membrane, Cell Culture, Selection, Fluorescence, Co-Culture Assay