Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Application of ADP prior to mechanical stimulation blocks ICWs. (B-D) Application of 10 μM UTP (B) , 10 μM UDP (C) and 10 μM UDP-glucose (D) have no inhibitory effects on ICW communication. (E) Pretreatment with 10 μM for 15 min MRS2395 partially inhibits ICWs. (F) Preincubation with 10 μM MRS2211 for 15 min suppresses ICW propagation. (G) Summary inhibitory effects of nucleotides on ICWs within 75 μm (n ≥ 88 cells for each condition from at least three independent experiments). All values are expressed as mean ± SD. * P < 0.05 and *** P < 0.001, one-way ANOVA, Scheffé post hoc test. N.S., no significant difference. (H) Gene expression of P2Y receptors in BV-2 microglia at mRNA level detected by RT-PCR. The ten lanes in the gel are as follows: P2Y 1,2,4,6,12,13,14 (experimental group with primers directed towards the P2YR mRNA); nuclease-free water (negative control); GAPDH (positive control) and marker (with a list of standardized DNA sequences from 100 bp to 800 bp). (I) Quantitative statistical results of RT-PCR normalized to GAPDH (n = 3). All values are expressed as mean ± SD. (J) Western-blot assay indicates expression of P2Y 12 receptor in BV-2 microglia. (K) Immunolocalization of P2Y 12 receptor on the membrane of BV-2 cells. Scale bar = 30 μm.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Marker, Western Blot

Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: ① A range of nucleotides, including ATP, ADP and UDP etc., are released into extracellular space in response to mechanical stimulus. ② P2Y 12/13 receptors localized on the membrane of neighboring cells can sense ATP/ADP released from the stimulated cell, then activate PLC/IP 3 /Ca 2+ signaling. ③ Upon sensing and responding to released ATP/ADP, Ca 2+ mobilization sequentially occur in neighboring cells, thus perform as intercellular calcium waves. ④ ATP cannot activate P2Y 12/13 receptors directly. Ecto-NTPase located on the plasma membrane could catalyze hydrolysis of ATP and generates ADP that predominantly activates P2Y 12/13 receptors.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques:

Journal:

Article Title: Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y 13 receptor-type

doi: 10.1038/sj.bjp.0705588

Figure Lengend Snippet: P2Y receptor mRNA expression and immunohistochemistry in HEK 293-N26 cells. (A) Subsequent to total RNA extraction and RT–PCR amplification with primers specific for distinct P2Y receptor cDNA fragments (P2Y13, 25 cycles; P2Y1–P2Y12, 35 cycles), cDNA products were analyzed by agarose gel (1.5%) electrophoresis. A representative gel with ethidium bromide-stained cDNA fragments is shown: P2Y1 (528 bp), P2Y4 (431 bp), P2Y6 (380 bp), P2Y11 (410) and P2Y13 (575 bp). P2Y2 and P2Y12 transcripts were not detectable. (B) Representative fluorescence image of HEK 293-N26 cells after immunocytochemical labeling of P2Y1 (a) and P2Y4 receptors (c), with rabbit anti-P2Y1 and P2Y4 receptor antibodies (scale bar, 20 μm). P2Y2 receptor immunoreactivities (b) could not be detected.

Article Snippet: After fixation, washing with Tris-buffered saline (TBS, 0.05 M; pH 7.6) and blocking with 5% fetal calf serum (FCS), the cells were incubated with the rabbit anti-P2Y receptor antibodies (anti-P2Y 1 : 1 : 1500, SmithKline Beecham Pharmaceuticals, Harlow, Essex, U.K.; anti-P2Y 2 : 1 : 1000, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, MD, U.S.A.; anti-P2Y 4 : 1 : 1000, Alomone Labs, Jerusalem, Israel) with 0.1% Triton X-100, 5% FCS in TBS for 12 h at 4°C.

Techniques: Expressing, Immunohistochemistry, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Fluorescence, Labeling

Journal: Stem Cells Translational Medicine

Article Title: Human cord blood‐derived regulatory T ‐cell therapy modulates the central and peripheral immune response after traumatic brain injury

doi: 10.1002/sctm.19-0444

Figure Lengend Snippet: Multicolor flow cytometry microglia/myeloid cell panel

Article Snippet: BV421 , P2Y12 , n/a , Alomone Labs , APR‐020‐F , Mediates microglia chemotaxis.

Techniques: Flow Cytometry, Marker, Chemotaxis Assay

Journal: Stem Cells Translational Medicine

Article Title: Human cord blood‐derived regulatory T ‐cell therapy modulates the central and peripheral immune response after traumatic brain injury

doi: 10.1002/sctm.19-0444

Figure Lengend Snippet: t‐SNE visualization of changes in microglia populations after CCI and treatment. A, Visualization of changes in cell clusters at 7 days post‐CCI (left) and at 30 days post‐CCI (right). At 7 days post‐CCI, there are clear differences between the sham and CCI plot density plots (top row); however, there are no clear differences between the CCI and Treg 24 hours plots. The antibody heat maps demonstrate that these CCI‐specific cell clusters are positive for CD45, CD11bc, and P2Y12, indicating that these cells are microglia. At 30 days post‐CCI, there are persistent visual differences between the sham and CCI density plots; furthermore, the antibody heat maps demonstrate that these cell clusters are also microglia. B, Detailed visualized of CCI‐specific cell clusters (black circles) at 30 days post‐CCI (top). Below, these cell clusters are visualized in more detail with the CD11bc antibody heat map. Within these clusters, there are clear differences between the sham and CCI plots, indicating that the CCI results in long term changes in microglia populations at 30 days after injury. Furthermore, there are clear differences between the CCI and Treg 24 hours groups, with fewer cells present in the treatment group. Replicates: 7d cohort, N = 8; 30d cohort, N = 6. CCI, controlled cortical impact; Treg, regulatory T cells; t‐SNE, t‐distributed stochastic neighbor embedding

Article Snippet: BV421 , P2Y12 , n/a , Alomone Labs , APR‐020‐F , Mediates microglia chemotaxis.

Techniques:

Journal: Stem Cells Translational Medicine

Article Title: Human cord blood‐derived regulatory T ‐cell therapy modulates the central and peripheral immune response after traumatic brain injury

doi: 10.1002/sctm.19-0444

Figure Lengend Snippet: Flow cytometric characterization and comparison of microglia in the contralateral (uninjured) and ipsilateral (injured) hemispheres after CCI and Treg therapy at 7 days post‐CCI and 30 days post‐CCI. A, Absolute microglia counts, measured as cells/mg of brain tissue, at 7 days post‐CCI. B‐D, MFI of CD45, CD11bc, and P2Y12 expression 7 days post‐CCI, respectively. E, Absolute microglia counts, measured as cells/mg of brain tissue, at 30 days post‐CCI. F‐H, MFI of CD45, CD11bc, and P2Y12 expression 30 days post‐CCI, respectively. Statistical significance between sham and CCI is indicated with # P ≤ .05, ## P ≤ .01, ### P ≤ .001, and #### P ≤ .0001. Replicates: 7d cohort, N = 8; 30d cohort, N = 6. Statistical significance between CCI and Treg is indicated with * P ≤ .05, ** P ≤ .01, *** P ≤ .001, and **** P ≤ 0.0001. CCI, controlled cortical impact; MFI, median fluorescent intensity; Treg, regulatory T cells

Article Snippet: BV421 , P2Y12 , n/a , Alomone Labs , APR‐020‐F , Mediates microglia chemotaxis.

Techniques: Expressing

Journal: STAR Protocols

Article Title: Protocol to quantify immune cell distribution from the vasculature to the glioma microenvironment on sequential immunofluorescence multiplex images

doi: 10.1016/j.xpro.2024.103079

Figure Lengend Snippet:

Article Snippet: P2RY12 , Atlas Antibodies , Cat# HPA014518 Polyclonal Dilution 1/1,000.

Techniques: Recombinant, Imaging, Software