Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 2. P2rx7 promoted the bone formation of MSCs. a The CFU-F in MSCs derived from the control and the P2rx7-/- MSCs. b Mineralized nodules formed by MSCs of the control and the P2rx7-/- mice were analyzed by alizarin red staining. c, d The expressions of osteogenesis marker genes in the control and the P2rx7-/- MSCs, as assessed by western blotting (c) and qPCR (d). e, f Transplants consisting of MSCs from control and P2rx7-/- mice mixed with HA/TCP were analyzed by H&E staining (e), and immunofluorescent staining of CD31 and ACAN (f), n = 5 per group. g Mineralized nodules formed by MSCs under osteogenic induction when exposed to 0, 40 nM or 100 nM BzATP were showed by alizarin red staining. h The expressions of osteogenic markers Runx2, ALP, and OCN in MSCs exposed to 0, 40 nM or 100 nM BzATP, as assessed by western blotting. i, j Transplants consisting of MSCs treated with 0, 40, 100 nM BzATP from control mice mixed with HA/TCP were analyzed by H&E staining (i), and immunofluorescent staining of CD31 and ACAN (j), n = 5 per group. Scale bar: e, f, i, j: 50 lm. e, i yellow, new bone; black, blood vessel; blue, new cartilage. Data was presented as mean ± SD. The data (a-d, g) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (a, b, e, f) and one-way ANOVA (d, g, i, j). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Derivative Assay, Control, Staining, Marker, Western Blot, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 3. P2rx7 controls mitochondrial dynamics in MSCs. a OCR, calculated basal respiration and ATP-linked respiration of the control and the P2rx7-/- MSCs. b ATP contents of the control and the P2rx7-/- MSCs. c ECAR, and calculated glycolysis, glycolytic capability and glycolytic reverse of the control and the P2rx7-/- MSCs. d TEM images of mitochondria (arrows) in the control and the P2rx7-/- MSCs (n = 20 per group). e DWM of the control and the P2rx7-/- MSCs was measured by JC-1 staining (polymer: red; monomer: green). f The expressions of genes Nd1 and mt-Cytb in the control and the P2rx7-/- MSCs that related to the mitochondrial mass in cells assessed by qPCR. g DWM of MSCs treated with different concentrations of BzATP was analyzed by JC-1 staining. Scale bar: d: 500 nm; e, g: 100 lm. Data was presented as mean ± SD. The data (a, b, e-g) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (a-f) and one-way ANOVA (g). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Control, Staining, Polymer, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 4. P2rx7 regulated mitochondrial fusion through Mfn1. a The mitochondria (top) labeled by Mitotracker Red and mitochondrial morphology (bottom) based on Mitochondrial Analyzer after grey processing, analyzed by Airyscan confocal images in the control and P2rx7-/- MSCs. b The expression of genes associated with mitochondrial dynamics in the control and the P2rx7-/- MSCs as assessed by western blotting. c Representative micrographs visualizing P2rx7 (arrows) on the mitochondria. d Reprehensive Airyscan confocal images of mitochondrial morphology (Mitotracker Red, top) and mitochondrial morphology (bottom) based on Mitochondrial Analyzer after grey processing in the MSCs treated with 0, 40 nM and 100 nM BzATP. e The expression of genes associated with mitochondrial dynamics in the MSCs treated with 0, 40 nM and 100 nM BzATP as assessed by western blotting. f Western blotting analysis of mitochondrial (left) and cytosolic (right) fractions of the MSCs treated with 40 nM and 100 nM BzATP. g IHC staining of Mfn1 and Opa1 proteins in the control and the P2rx7-/- MSCs that were subcutaneously implanted into immunocompromised mice with HA/TCP (n = 5 per group). Scale bar: a, d:10 lm (top), 5 lm(bottom); g: 100 lm; h: 500 lm. Data was presented as mean ± SD. The data (a, b, d-f) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Labeling, Control, Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 5. P2rx7 enhanced the sensitivity of ERK pathway to promote mitochondrial fusion. a Top 10 significantly enrichment of downregulated DEGs between control and P2rx7-/- MSCs analyzed by KEGG. b Expressions of Erk1/2 and p-Erk1/2 protein in the distal femurs were analyzed by IHC staining (n = 5 per group). c The phosphorylation level and the total expression level of Erk1/2 in the control and the P2rx7-/- MSCs before and after the activated by bFGF, as assessed by western blotting. d The phosphorylation level and the total expression level of Erk1/2 in MSCs treated with different concentrations of BzATP before and after the activated by 10 lM bFGF for 1 h, as assessed by western blotting. e Expressions of genes associated with mitochondrial dynamics in the Widetype (WT) and the P2rx7-/- MSCs treated with bFGF as assessed by western blotting. f Expressions of genes associated with mitochondrial dynamics in MSCs treated with siNC, siMfn1 and BzATP as assessed by western blotting. g DWM of the Widetype (WT) and the P2rx7-/-MSCs treated with BzATP and bFGF was analyzed by JC-1 staining. h ALP activity (top) and mineralized nodules (bottom) under osteogenic induction of human MSCs when exposed to 100 nM BzATP in the presence and absence of U0126. i The expression of osteogenesis marker genes in MSCs exposed with BzATP, in the presence and absence of 10 lM U0126. Scale bar: b: 100 lm; d: 50 lm; h: 500 lm. Data was presented as mean ± SD. The data (c-i) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (b) and one-way ANOVA (g, h).

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Control, Immunohistochemistry, Phospho-proteomics, Expressing, Western Blot, Staining, Activity Assay, Marker, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 6. Bone metabolism and regeneration could be promoted by DCA treatment. a, b Increased mineralized nodules (a) and ALP activity (b) under osteogenic induction of the P2rx7-/- MSCs treated with PBS (Control) and 5 lM DCA. c, d Expressions of osteogenesis marker genes in the P2rx7-/- MSCs treated with Control and 5 lM DCA, as assessed by western blotting (c) and qPCR analysis (d). e, f Cross-sectional lCT images of the trabecular bone (e) and H&E staining of distal femurs (f) of 4-month-old P2rx7-/- mice treated with and without DCA, n = 5 per group. g, h Mouse mandibles of the Control and the BzATP group after 4 weeks of healing were presented as lCT images (g) and statistical analysis (g right, BV/TV in lCT images; h right, percent of area of bone tissue according to Masson staining), H&E staining (h, top) and Masson staining (h, bottom), n = 5 per group. Black arrow: new bone. i Expressions of Mfn1 and Opa1 proteins in the area of defect after 4-week healing were analyzed by IHC staining (n = 5 per group). Scale bar: f, h: 500 lm; i: 100 lm. Data was presented as mean ± SD. The data (a-d) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two- tailed Student’s t test.

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Activity Assay, Control, Marker, Western Blot, Staining, Immunohistochemistry, Two Tailed Test

Journal: Experimental Eye Research

Article Title: Effects of Cardiotonic Steroids on Trabecular Meshwork Cells: Search for Mediator of Ouabain-Enhanced Outflow Facility

doi: 10.1016/j.exer.2012.01.009

Figure Lengend Snippet: TM5 cells with ouabain (OUA, 30 nM and 100 nM, 4 hrs) exposure displayed similar expression levels of PX1, Cx43, P2RX7, A1 adenosine receptors (ADOR1), and β-actin (ACTB) genes, compared with non-treated (NT) cells (P>0.05 by one-way ANOVA). Data obtained from 5 independent experiments were normalized to PX1 gene of NT Group, using GAPDH as loading control.

Article Snippet: P2RX 7 , Hs00175721_m1.

Techniques: Expressing, Control

Journal: Journal of Bone Oncology

Article Title: The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties

doi: 10.1016/j.jbo.2021.100398

Figure Lengend Snippet: Characterising P2RX7B in TE85 and MNNG-HOS OS cells: A) Expression of P2RX7 mRNA in TE85, TE85 + P2RX7B, MNNG-HOS and MNNG-HOS + P2RX7B cells with two different P2RX7 sets of primers binding to the P2RX7 N Terminal or C terminal, (product length 413 BP and 399 BP respectively) HEK-293 + P2RX7A was used as a positive control. B) Expression of P2RX7 mRNA in TE85, TE85 + P2RX7B, MNNG-HOS and MNNG-HOS + P2RX7B OS cells using qRT-PCR, HPRT was used as a housekeeping gene. C) Calcium assays were assessed in TE85, TE85 + P2RX7B, MNNG-HOS and MNNG-HOS + P2RX7B cells by loading 15,000 cells with Fluo-4 Direct™ calcium reagent, left to incubate for 1 h at 37 °C before been stimulated with 100 µM BzATP and then 0.8 µM ionomycin, the response was detected at excitation ratio and emission wavelength 490 nm and 525 nm respectively for five minutes. D) Measurement of plasma membrane permeabilization was performed on TE85, TE85 + P2RX7B, MNNG-HOS and MNNG-HOS + P2RX7B cells using ethidium bromide uptake, 15,000 cells in HBSS were incubated at 37 °C with or without 10 μM of the P2RX7 inhibitor A740003 for 1 h. BzATP was diluted in ultra-pure ethidium bromide to a final concentration of 300 μM BzATP and 100 μM ethidium bromide, the response was detected at 360 nm excitation and 580 nm emission for 45 min after an initial 5-minute baseline reading, with readings taken every 2 min, HEK-293 + P2RX7A was used as a positive control. All data is from 3 biological repeats with 6 technical replicates per experiment and were compared using an unpaired T-test, * = P < 0.05 ** = P < 0.01.

Article Snippet: qRT-PCR was performed using Taqman probes (Human P2RX7 Taqman® gene expression assay, ID: Hs00951600_m1 catalogue: 4351372, human HPRT Taqman® gene expression assay, ID: Hs02800695 catalogue: 1621448) in accordance with the manufacturer’s instructions on a Applied Biosystems 7900HT Real-Time PCR machine (Applied BiosystemsTM).

Techniques: Expressing, Binding Assay, Positive Control, Quantitative RT-PCR, Clinical Proteomics, Membrane, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X7R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X7R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with C57BL/6J mice. Student’s t -test * p < 0.05, ** p < 0.01. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40–P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retinas analyzed by immunohistochemistry. Cross-sectional cryosections of retinas from a representative C57BL/6J mouse ( A , D , G ) and rd10early ( B , E , H ) and rd10late ( C , F , I ) mice, stained with antibodies against P2X7R ( A – C , G – I ). Nuclei were stained with TO-PRO ( D – F ). In C57BL/6J mice, immunopositive fluorescence against P2X7R appears mainly located in the INL and GCL. RPE: retinal pigment epithelial cells; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 µm.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X4R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X4R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X4R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with the control expression in C57BL/6J mice. Student’s t -test, * p < 0.05. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40-P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in retinal myeloid cells (CD11b + ). The CD11b immunoreactive cell population was analyzed by flow cytometry. Bar graphs show the number of CD11b + cells expressing P2X7R ( A ) and the mean intensity of P2X7R fluorescence values ( B ). Also, the number of CD11b + cells expressing P2X4R is shown ( C ), and the mean intensity of P2X4R fluorescence values ( D ). C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44. ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in the CD11b + population, analyzed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b + cells were gated ( A ) and the expression of P2X7R and P2X4R was analyzed ( B – D ). ( B ) Double contour plots representing P2X7R and P2X4R expression, showing P2X7R- and P2X4R-positive populations (green line) and P2X7R- and P2X4R-highly immunoreactive populations (blue line). Each plot shows the sum of a minimum of 3 independent replicates. ( C , D ) Bar graphs showing the number of double-positive cells (P2X7R and P2X4R) and mean fluorescence values for P2X7R and P2X4R in the whole double-positive population ( C ) and the highly immunoreactive double-positive population ( D ). Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-statistically significant. C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression analysis in CD11b+ cells, assessed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b-immunopositive cells were gated and two populations were selected according to medium or high immunoreactivity against the CD11b antibody, as shown in the contour plot (rd10 postnatal day [P]18) ( A ). The dot plot shows the sum of a minimum of 3 independent replicates. The immunoreactivity against P2X7R ( B ) and P2X4R ( C ) was compared in the two CD11b populations of each group of mice. Student’s t -test was performed between the two populations in each condition. Data are presented as mean values ± SD. Student’s t -test, ** p < 0.01. C57BL/6J: P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining