flow cytometry antibody conjugated with allophycocyanin  (Miltenyi Biotec)

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: Differential roles of P2X4 and P2X7 receptors in inducing sEV production under hypoxic conditions. CTL, CR4 (knock-down for the expression of P2X4) and CR7 (knock-down for the expression of P2X7) cells were grown under hypoxic conditions (1% O2) for a duration of 48 h prior to conducting analyses. a Size distribution and concentration of EVs purified from CTL, CR4 or CR7 cells grown under hypoxia, assessed by NTA (n = 12 independent experiments for CTL and CR4, n = 6 independent experiments for CR7). b Relative concentrations of sEVs per mL of supernatants, expressed relatively to that for CTL, in the same conditions than in a. The concentration of sEV produced by CR4 cells was significantly higher as compared to CTL (**, p <0.01). NS stands for not statistically different. c Protein concentrations of sEV extracts from same conditions than in a. There was a significantly higher protein concentration of sEVs extracts from CR4 cells, as compared to CTL (*, p <0.05) or to CR7 (*, p <0.05) cells. NS stands for not statistically different. d Relative concentration of sEVs per mL of supernatants coming from CTL cells treated with either 5-BDBD (P2X4 antagonist, 5µM) or A438079 (P2X7 antagonist, 10 µM) and expressed relatively to the CTL condition (vehicle). There was a significantly higher concentration of sEV produced by cells submitted to the P2X4 antagonist as compared to CTL (*, p <0.05) but not with cells treated with the P2X7 antagonist. NS stands for not statistically different. e Mean size (in nm) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in same conditions than in a. EVs produced by CR4 cells were significantly smaller than CTL (*, p <0.05). NS stands for not statistically different. f Zeta potential (in mV) of sEVs purified from supernatants coming from CTL, CR4 or CR7 cells, in similar conditions than in a. There was no difference between the three groups (n = 4 independent experiments). g Proportion of CD9+, CD63+ and CD81+ sEV purified from CR4 cells supernatants, assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 12 independent experiments). There was a significantly higher proportion of CD9+ sEV produced by CR4 cells compared to CTL cells (*, p <0.05). h Proportion of CD9+, CD63+ and CD81+ sEVs purified from supernatants of CTL cells treated with 5-BDBD (P2X4 antagonist, 5 µM), assessed by Fluo-NTA, normalized to that coming from CTL cells (n = 8 independent experiments). There was a significantly higher proportion of CD9+ sEVs produced by cells treated with 5-BDBD (*, p <0.05). i Proportion of CD9+, CD63+ and CD81+ sEVs purified from CR7 cells, assessed by Fluo-NTA, normalized to that from CTL cells (n = 5 independent experiments). There was no statistical difference. j Proportion of CD9+-, CD63+- and CD81+-sEVs purified from CTL cells treated with A438079 (P2X7 antagonist, 10 µM), assessed by Fluo-NTA, normalized to that from CTL cells (n = 4 independent experiments). There was no statistical difference. k Intracellular expression of tetraspanins CD9, CD63, CD81 in CTL, CR4 and CR7 cells analysed by flow cytometry. Data are represented in relative MFI with mean ± SD normalized to that in 4T1 cells in CTL condition (n = 8). (**, p < 0.01). NS stands for no statistical difference. l Representative western blot showing the protein content and sEV production in CTL, CR4 and CR7 cells, under both normoxia and hypoxia. Markers of sEVs (CD9, CD63, CD81, TSG101, Flotillin1, Syntinin-1), are enriched in extracellular vesicle fractions, but not GRP94 used as a marker of endoplasmic reticulum which was only identified in total cell protein extract. sEVs produced by CR4 cells demonstrated a higher level of CD9. HSC70 and β-actin were used as control proteins

Article Snippet: For CD63 staining, we used a flow cytometry antibody conjugated with allophycocyanin (APC, Miltenyi Biotec, France, 130-123-276), for 2 h at room temperature in the dark.

Techniques: Knockdown, Expressing, Concentration Assay, Purification, Produced, Protein Concentration, Zeta Potential Analyzer, Flow Cytometry, Western Blot, Marker, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The P2X4 purinergic receptor controls the autophagy-related release of small extracellular vesicles from mammary cancer cells under hypoxia

doi: 10.1186/s12964-026-02811-5

Figure Lengend Snippet: sEVs produced from P2X4-expressing cancer cells enhance the invasive capacities of recipient cancer cells. a CTL cancer cells were treated with EVs purified from CTL, CR4 or CR7 cells, grown under hypoxic conditions (1% O2). EVs were stained with green, fluorescent membrane probe PKH67. Representative fluorescent images obtained after 4 h treatment with 20 µg/mL of EVs. Upper, merged images showing DAPI staining for identification of cell nuclei and PKH67 staining showing EVs. Lower, PKH67 staining alone. White arrows indicate EVs incorporated into CTL recipient cells. Scale bars, 75 μm. b Assessment of the uptake as a function of time of PKH67-stained EVs, produced by CTL, CR4, CR7 cells, in CTL cancer recipient cells by flow cytometry (treatment with 20 µg/mL EVs protein). Results indicate a speed of uptake expressed as a ratio of the uptake after 4-h on that after 1-h incubation. Data are represented in rMFI with Mean MFI 4 h normalized to their respective control MFI 1 h, mean ± SD (n = 6 independent experiments). *, indicate a statistical difference at p < 0.05. NS stands for no statistical difference. c Cartoon of the experimental procedure of co-culture assays to assess cell invasiveness performed in hypoxic conditions (1% O2). Invasiveness of recipient CTL was assessed as being their capacity to invade an 8 μm-sized filter covered by a thin layer of Matrigel. These cells were co-cultured with CTL, CR4, CR7 donor cells seeded at the bottom of the well. d Invasive capacities of recipient CTL cells co-cultured with CTL, CR4 or CR7 donor cells, as illustrated in c. Experiments were performed in the absence (vehicle, DMSO) or presence of the nSMAse inhibitor GW4869 (10 µM). Data are normalized to the control condition, in absence of GW4869, and presented as mean ± SD ( n = 6 independent experiments). *, p < 0.05 and **, p < 0.01. NS stands for no statistical difference

Article Snippet: For CD63 staining, we used a flow cytometry antibody conjugated with allophycocyanin (APC, Miltenyi Biotec, France, 130-123-276), for 2 h at room temperature in the dark.

Techniques: Produced, Expressing, Purification, Staining, Membrane, Flow Cytometry, Incubation, Control, Co-Culture Assay, Cell Culture

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Staining, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Adoptive Transfer Assay, Transfection, Control, Infection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Binding Assay, Derivative Assay, Activation Assay, Expressing, In Vivo