ionomycin  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    Alomone Labs ionomycin
    Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to <t>ionomycin</t> stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p
    Ionomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Alomone Labs
    Average 90 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2022-08
    90/100 stars

    Images

    1) Product Images from "Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion"

    Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009022

    Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p
    Figure Legend Snippet: Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p

    Techniques Used: Activity Assay, Functional Assay, Infection, Fluorescence, Microscopy, Expressing, Staining, Whisker Assay

    Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion. (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p
    Figure Legend Snippet: Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion. (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p

    Techniques Used: Inhibition, Activity Assay, Expressing, Cell Culture, Fluorescence, Microscopy, Translocation Assay, Activation Assay, Staining, Binding Assay, Western Blot, Transfection, Whisker Assay

    2) Product Images from "A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1"

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.115.136085

    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P
    Figure Legend Snippet: Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Techniques Used: Labeling, Fluorescence

    [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P
    Figure Legend Snippet: [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Techniques Used: Fluorescence, Size-exclusion Chromatography, Mouse Assay

    Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.
    Figure Legend Snippet: Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Techniques Used: Fluorescence, Labeling

    Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P
    Figure Legend Snippet: Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Techniques Used: Mouse Assay

    Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P
    Figure Legend Snippet: Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Techniques Used: Mouse Assay, Sequencing, Fluorescence

    3) Product Images from "Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals"

    Article Title: Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals

    Journal: Neuron

    doi: 10.1016/j.neuron.2017.01.010

    Presynaptic inhibition is slower than ER Ca 2+ depletion following SERCA block (A-C) Average axonal ER Ca 2+ dynamics were measured using ER-GCaMP6-150 at 26°C, (A, blue) or 37°C (B, red). Grey traces are individual experiments, although some individual responses overshot the scale used here and their peaks were not included in the graph. Neurons were stimulated with 20 AP (20 Hz) and then treated with CPA to induce ER Ca 2+ depletion. After 3 min of CPA treatment the responses to a second stimulus were abolished (indicated by second arrow, 20AP 20Hz). (C) Estimate of Δ[Ca 2+ ] ER assuming an average resting [Ca 2+ ] ER based on ionomycin responses; n(26°C)=8, n(37°C)=15; n.s. p=0.36. (D) Single-AP cytosolic Ca 2+ responses (Fluo-5F AM, normalized to average pre-CPA ΔF response) every 60s measured before (black traces) and after CPA treatment at 26°C (D, G, blue traces) or 37°C (E, H, red traces). (G, H) Comparison of the kinetics of presynaptic inhibition and axonal ER Ca 2+ depletion. Curves were fit to single exponential decays where possible and time constants (τ) were obtained for comparison. τ ER (26°C)= 47.5 ± 3.4 s, n=8; τ ER (37°C)= 26 ± 0.9 s, n=15; τ cyto Ca 2+ (37°C)= 148 ± 18 s, n=7.
    Figure Legend Snippet: Presynaptic inhibition is slower than ER Ca 2+ depletion following SERCA block (A-C) Average axonal ER Ca 2+ dynamics were measured using ER-GCaMP6-150 at 26°C, (A, blue) or 37°C (B, red). Grey traces are individual experiments, although some individual responses overshot the scale used here and their peaks were not included in the graph. Neurons were stimulated with 20 AP (20 Hz) and then treated with CPA to induce ER Ca 2+ depletion. After 3 min of CPA treatment the responses to a second stimulus were abolished (indicated by second arrow, 20AP 20Hz). (C) Estimate of Δ[Ca 2+ ] ER assuming an average resting [Ca 2+ ] ER based on ionomycin responses; n(26°C)=8, n(37°C)=15; n.s. p=0.36. (D) Single-AP cytosolic Ca 2+ responses (Fluo-5F AM, normalized to average pre-CPA ΔF response) every 60s measured before (black traces) and after CPA treatment at 26°C (D, G, blue traces) or 37°C (E, H, red traces). (G, H) Comparison of the kinetics of presynaptic inhibition and axonal ER Ca 2+ depletion. Curves were fit to single exponential decays where possible and time constants (τ) were obtained for comparison. τ ER (26°C)= 47.5 ± 3.4 s, n=8; τ ER (37°C)= 26 ± 0.9 s, n=15; τ cyto Ca 2+ (37°C)= 148 ± 18 s, n=7.

    Techniques Used: Inhibition, Blocking Assay

    4) Product Images from "A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1"

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.115.136085

    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P
    Figure Legend Snippet: Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Techniques Used: Labeling, Fluorescence

    [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P
    Figure Legend Snippet: [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Techniques Used: Fluorescence, Mouse Assay

    Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.
    Figure Legend Snippet: Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Techniques Used: Fluorescence, Labeling

    Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P
    Figure Legend Snippet: Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Techniques Used: Mouse Assay

    Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P
    Figure Legend Snippet: Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Techniques Used: Mouse Assay, Sequencing, Fluorescence

    5) Product Images from "SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System"

    Article Title: SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System

    Journal: Stem Cell Reviews and Reports

    doi: 10.1007/s12015-021-10179-x

    iSOX9-astrocytes show comparable calcium responses as fHA. Calcium responses (plotted as F340/F380 ratio) measured in Fura-2-loaded iSOX9-astrocytes and fHA in the presence of extracellular Ca 2+ -chelating agent BAPTA (3 mM), ensuring only Ca 2+ release from internal Ca 2+ stores is measured. a After stimulating fHA and iSOX9-astrocytes with ATP, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). b After stimulating fHA and iSOX9-astrocytes with Ach, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). c iSOX9-astrocytes and fHA show a similar calcium response after stimulation with GPN, FCCP and ionomycin measured by Fura2 fluorescence emission. A significantly higher calcium response is observed in fHA compared to iSOX9-astrocytes after thapsigargin stimulation (**p
    Figure Legend Snippet: iSOX9-astrocytes show comparable calcium responses as fHA. Calcium responses (plotted as F340/F380 ratio) measured in Fura-2-loaded iSOX9-astrocytes and fHA in the presence of extracellular Ca 2+ -chelating agent BAPTA (3 mM), ensuring only Ca 2+ release from internal Ca 2+ stores is measured. a After stimulating fHA and iSOX9-astrocytes with ATP, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). b After stimulating fHA and iSOX9-astrocytes with Ach, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). c iSOX9-astrocytes and fHA show a similar calcium response after stimulation with GPN, FCCP and ionomycin measured by Fura2 fluorescence emission. A significantly higher calcium response is observed in fHA compared to iSOX9-astrocytes after thapsigargin stimulation (**p

    Techniques Used: Fluorescence

    6) Product Images from "Acrosome Reaction and Ca2+ Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2+]i 1"

    Article Title: Acrosome Reaction and Ca2+ Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2+]i 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.114.119768

    AR and [Ca 2+ ]i imaging in single human spermatozoa exposed to ionomycin and progesterone. A ) Nonstimulated human spermatozoa labeled with FM4-64 (red) and Fluo-4 (green). FM4-64 labels the plasma membrane, and Fluo-4 is distributed mainly in the cytoplasm.
    Figure Legend Snippet: AR and [Ca 2+ ]i imaging in single human spermatozoa exposed to ionomycin and progesterone. A ) Nonstimulated human spermatozoa labeled with FM4-64 (red) and Fluo-4 (green). FM4-64 labels the plasma membrane, and Fluo-4 is distributed mainly in the cytoplasm.

    Techniques Used: Imaging, Labeling

    Evidence that FM4-64 acrosome staining corresponds to the onset of the AR. A ) Concomitant imaging of FM4-64 and FITC-PSA during ionomycin application to human spermatozoa. Ionomycin applied at ∼80 sec (black arrowhead indicates its addition) increases
    Figure Legend Snippet: Evidence that FM4-64 acrosome staining corresponds to the onset of the AR. A ) Concomitant imaging of FM4-64 and FITC-PSA during ionomycin application to human spermatozoa. Ionomycin applied at ∼80 sec (black arrowhead indicates its addition) increases

    Techniques Used: Staining, Imaging, Size-exclusion Chromatography

    7) Product Images from "Balance of calcineurin A? and CDK5 activities sets release probability at nerve terminals"

    Article Title: Balance of calcineurin A? and CDK5 activities sets release probability at nerve terminals

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.4288-12.2013

    CNAα and CDK5 gate silent presynaptic boutons by controlling Ca 2+ influx (A) Representative single-AP GCaMP3 difference image (ΔF 1AP ) in WT neurons in the absence or presence of CSA (Top) or Ros (Bottom). Ionomycin treatment (right) reveals all presynaptic boutons. White circles indicate boutons that were silenced after treatment with CSA (Top) or unsilenced after treatment of Ros (Bottom). Scale bar represents 5 μm. (B) Corresponding traces of silenced (white circle in A top) and unsilenced boutons (white circle in A bottom) with or without treatment of CSA or Ros. (C) Histogram of the fraction of silent and responsive boutons measured by GCaMP3 in WT with or without CSA or Roscovitine: WT silent = 0.17 ± 0.04, WT responsive = 0.83 ± 0.04 (n= 25), WT+CSA silent = 0.46 ± 0.09, WT+CSA responsive = 0.54 ± 0.09 (n= 9), WT+Ros silent = 0.07 ± 0.02, WT+Ros responsive = 0.93 ± 0.02 (n= 16). (D) Histogram of the proportion of silent and responsive boutons measured by MgGreen in WT with or without CSA or Ros. The division of silent or responsive boutons was based on a signal to noise ratio of 1 (peak versus SD of baseline) for the single-AP response: WT silent = 0.14 ± 0.03, WT responsive = 0.86 ± 0.03 (n=19), WT+CSA silent = 0.36 ± 0.11, WT+CSA responsive = 0.64 ± 0.11 (n=7), WT+Ros silent = 0.04 ± 0.02, WT+Ros responsive = 0.96 ± 0.02 (n=10). (E) Histogram of the fraction of silent boutons measured by GCaMP3 and normalized to fraction of WT silent boutons in WT, CNAαKD, CDK5KD, WT with/without CSA or Roscovitine, WT with Cono or Aga with/without Roscovitine.: WT = 1.0 ± 0.18 (n=25), CNAαKD = 3.00 ± 0.24 (n=26), CDK5KD = 0.36 ± 0.09 (n= 12), WT+CSA = 2.70 ± 0.54 (n= 9), WT+Ros = 0.40 ± 0.09 (n= 16), WT+Cono = 3.31 ± 0.66, WT+Cono +Ros = 2.85 ± 0.60 (n= 7), WT+Aga = 1.87 ± 0.16, WT+Aga+Ros = 0.76 ± 0.40 (n= 8).
    Figure Legend Snippet: CNAα and CDK5 gate silent presynaptic boutons by controlling Ca 2+ influx (A) Representative single-AP GCaMP3 difference image (ΔF 1AP ) in WT neurons in the absence or presence of CSA (Top) or Ros (Bottom). Ionomycin treatment (right) reveals all presynaptic boutons. White circles indicate boutons that were silenced after treatment with CSA (Top) or unsilenced after treatment of Ros (Bottom). Scale bar represents 5 μm. (B) Corresponding traces of silenced (white circle in A top) and unsilenced boutons (white circle in A bottom) with or without treatment of CSA or Ros. (C) Histogram of the fraction of silent and responsive boutons measured by GCaMP3 in WT with or without CSA or Roscovitine: WT silent = 0.17 ± 0.04, WT responsive = 0.83 ± 0.04 (n= 25), WT+CSA silent = 0.46 ± 0.09, WT+CSA responsive = 0.54 ± 0.09 (n= 9), WT+Ros silent = 0.07 ± 0.02, WT+Ros responsive = 0.93 ± 0.02 (n= 16). (D) Histogram of the proportion of silent and responsive boutons measured by MgGreen in WT with or without CSA or Ros. The division of silent or responsive boutons was based on a signal to noise ratio of 1 (peak versus SD of baseline) for the single-AP response: WT silent = 0.14 ± 0.03, WT responsive = 0.86 ± 0.03 (n=19), WT+CSA silent = 0.36 ± 0.11, WT+CSA responsive = 0.64 ± 0.11 (n=7), WT+Ros silent = 0.04 ± 0.02, WT+Ros responsive = 0.96 ± 0.02 (n=10). (E) Histogram of the fraction of silent boutons measured by GCaMP3 and normalized to fraction of WT silent boutons in WT, CNAαKD, CDK5KD, WT with/without CSA or Roscovitine, WT with Cono or Aga with/without Roscovitine.: WT = 1.0 ± 0.18 (n=25), CNAαKD = 3.00 ± 0.24 (n=26), CDK5KD = 0.36 ± 0.09 (n= 12), WT+CSA = 2.70 ± 0.54 (n= 9), WT+Ros = 0.40 ± 0.09 (n= 16), WT+Cono = 3.31 ± 0.66, WT+Cono +Ros = 2.85 ± 0.60 (n= 7), WT+Aga = 1.87 ± 0.16, WT+Aga+Ros = 0.76 ± 0.40 (n= 8).

    Techniques Used:

    8) Product Images from "Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm"

    Article Title: Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22010093

    Qualitative evaluation of progesterone (Pg)-induced intracellular calcium concentration [Ca 2+ ] i changes in human sperm. ( a ) Representative time-lapse flow cytometry (TLFC) Fluo3-AM (Fluo3) fluorescence density plot of a sperm sample from a known paternity donor under capacitation conditions. Gray boxes above the plot indicate the nature and duration of the employed stimuli: progesterone (Pg), ionomycin (Iono), and manganese chloride (Mn 2+ ). The dark solid line indicates the median fluorescence value. ( b ) Representative example of normalized median fluorescence values ((F − F 0 )/F 0 ) for an entire sperm population incubated under non-capacitating (NC) or capacitating (CAP) conditions. The F Peak is indicated for both traces. Gray boxes above the plot indicate the nature and duration of the employed stimuli. ( c ) Comparison of F Peak responses for individual sperm samples (shown color-coded) from known ( n = 7) and no-known ( n = 11) paternity donors, incubated either under NC or CAP conditions. Black dots and solid lines indicate mean ± SEM; * p
    Figure Legend Snippet: Qualitative evaluation of progesterone (Pg)-induced intracellular calcium concentration [Ca 2+ ] i changes in human sperm. ( a ) Representative time-lapse flow cytometry (TLFC) Fluo3-AM (Fluo3) fluorescence density plot of a sperm sample from a known paternity donor under capacitation conditions. Gray boxes above the plot indicate the nature and duration of the employed stimuli: progesterone (Pg), ionomycin (Iono), and manganese chloride (Mn 2+ ). The dark solid line indicates the median fluorescence value. ( b ) Representative example of normalized median fluorescence values ((F − F 0 )/F 0 ) for an entire sperm population incubated under non-capacitating (NC) or capacitating (CAP) conditions. The F Peak is indicated for both traces. Gray boxes above the plot indicate the nature and duration of the employed stimuli. ( c ) Comparison of F Peak responses for individual sperm samples (shown color-coded) from known ( n = 7) and no-known ( n = 11) paternity donors, incubated either under NC or CAP conditions. Black dots and solid lines indicate mean ± SEM; * p

    Techniques Used: Concentration Assay, Flow Cytometry, Fluorescence, Incubation

    9) Product Images from "Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis"

    Article Title: Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-021-00894-w

    Bcl-xL overexpression inhibits IP 3 R-mediated Ca 2+ release in living cells. Ca 2+ signals were measured in Fura-2-loaded HEK cells expressing empty vector (pCMV24-P2A-mCherry; black), Bcl-2 (pCMV24-3xFLAG-Bcl-2-P2A-mCherry; orange) or Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green). EGTA (3 mM) was added to chelate extracellular Ca 2+ . IP 3 R-mediated Ca 2+ release was evoked by ATP (10 µM) ( a – e ) or carbachol (Cch, 10 µM) ( f – j ). Ionomycin (iono, 5 µM diluted in 250 mM CaCl 2 ) was added to assess the maximal Ca 2+ response. Representative single-cell Ca 2+ responses obtained from one well containing about 20–40 cells are shown ( a – c , f – h ). For each condition, six to nine different wells obtained from two to three independent transfections were monitored. Percentages of responding cells (d , i ) and areas under the curve ( e , j ) were calculated from the Ca 2+ traces. Data are represented as mean of wells ± SD ( N = 6–9), each data point represents one well. Statistically significant differences were determined using a one-way ANOVA (* P
    Figure Legend Snippet: Bcl-xL overexpression inhibits IP 3 R-mediated Ca 2+ release in living cells. Ca 2+ signals were measured in Fura-2-loaded HEK cells expressing empty vector (pCMV24-P2A-mCherry; black), Bcl-2 (pCMV24-3xFLAG-Bcl-2-P2A-mCherry; orange) or Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green). EGTA (3 mM) was added to chelate extracellular Ca 2+ . IP 3 R-mediated Ca 2+ release was evoked by ATP (10 µM) ( a – e ) or carbachol (Cch, 10 µM) ( f – j ). Ionomycin (iono, 5 µM diluted in 250 mM CaCl 2 ) was added to assess the maximal Ca 2+ response. Representative single-cell Ca 2+ responses obtained from one well containing about 20–40 cells are shown ( a – c , f – h ). For each condition, six to nine different wells obtained from two to three independent transfections were monitored. Percentages of responding cells (d , i ) and areas under the curve ( e , j ) were calculated from the Ca 2+ traces. Data are represented as mean of wells ± SD ( N = 6–9), each data point represents one well. Statistically significant differences were determined using a one-way ANOVA (* P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Transfection

    Bcl-xL, but not Bcl-xL K87D , overexpression inhibits IP 3 R-mediated Ca 2+ release in single cells. Calcium measurements obtained from Fura-2-loaded HeLa ( a–c ) and COS-7 cells ( d–g ) transfected with a Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green) or Bcl-xL K87D -coding vector (pCMV24-3xFLAG-Bcl-xL K87D -P2A-mCherry; red), or an empty vector (pCMV24-P2A-mCherry; black). ER Ca 2+ response is elicited by addition of 70 nM (HeLa) or 500 nM (Cos-7) ATP, after addition of 3 mM EGTA to chelate extracellular Ca 2+ . Ionomycin (5 µM) diluted in 250 mM CaCl 2 was added at the end of the experiment (not shown) to trigger a high Ca 2+ release and confirm all the cells are equally loaded with Fura-2. For each condition, three to five wells were monitored and about 20–30 cells were analyzed by well. a Representative traces of Ca 2+ release in COS-7 cells. Traces represent mean ± SEM of one representative measurement (one well, about 20–30 cells). Percentage of responding cells ( b ) and maximal peak amplitude ( c ) were calculated for each condition. Data represent mean ± SD of four independent experiments ( N = 4). Statistically significant differences were determined using a one-way ANOVA (* P
    Figure Legend Snippet: Bcl-xL, but not Bcl-xL K87D , overexpression inhibits IP 3 R-mediated Ca 2+ release in single cells. Calcium measurements obtained from Fura-2-loaded HeLa ( a–c ) and COS-7 cells ( d–g ) transfected with a Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green) or Bcl-xL K87D -coding vector (pCMV24-3xFLAG-Bcl-xL K87D -P2A-mCherry; red), or an empty vector (pCMV24-P2A-mCherry; black). ER Ca 2+ response is elicited by addition of 70 nM (HeLa) or 500 nM (Cos-7) ATP, after addition of 3 mM EGTA to chelate extracellular Ca 2+ . Ionomycin (5 µM) diluted in 250 mM CaCl 2 was added at the end of the experiment (not shown) to trigger a high Ca 2+ release and confirm all the cells are equally loaded with Fura-2. For each condition, three to five wells were monitored and about 20–30 cells were analyzed by well. a Representative traces of Ca 2+ release in COS-7 cells. Traces represent mean ± SEM of one representative measurement (one well, about 20–30 cells). Percentage of responding cells ( b ) and maximal peak amplitude ( c ) were calculated for each condition. Data represent mean ± SD of four independent experiments ( N = 4). Statistically significant differences were determined using a one-way ANOVA (* P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    10) Product Images from "Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis"

    Article Title: Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.218958

    Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).
    Figure Legend Snippet: Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).

    Techniques Used: Imaging, Microscopy, Fluorescence

    Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10†µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .
    Figure Legend Snippet: Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10†µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .

    Techniques Used: Labeling, Sequencing, Fluorescence

    11) Product Images from "Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis"

    Article Title: Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.218958

    Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).
    Figure Legend Snippet: Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).

    Techniques Used: Imaging, Microscopy, Fluorescence

    Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10 µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .
    Figure Legend Snippet: Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10 µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .

    Techniques Used: Labeling, Sequencing, Fluorescence

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Alomone Labs ionomycin
    Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to <t>ionomycin</t> stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p
    Ionomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Alomone Labs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2022-08
    90/100 stars
      Buy from Supplier

    Image Search Results


    Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p

    Journal: PLoS Pathogens

    Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

    doi: 10.1371/journal.ppat.1009022

    Figure Lengend Snippet: Calcineurin phosphatase activity remains functional in VZV infected cells. (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p

    Article Snippet: Validation of compounds from HT-SRFA Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich).

    Techniques: Activity Assay, Functional Assay, Infection, Fluorescence, Microscopy, Expressing, Staining, Whisker Assay

    Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion. (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p

    Journal: PLoS Pathogens

    Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

    doi: 10.1371/journal.ppat.1009022

    Figure Lengend Snippet: Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion. (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p

    Article Snippet: Validation of compounds from HT-SRFA Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich).

    Techniques: Inhibition, Activity Assay, Expressing, Cell Culture, Fluorescence, Microscopy, Translocation Assay, Activation Assay, Staining, Binding Assay, Western Blot, Transfection, Whisker Assay

    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Labeling, Fluorescence

    [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Fluorescence, Size-exclusion Chromatography, Mouse Assay

    Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Fluorescence, Labeling

    Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Mouse Assay

    Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Mouse Assay, Sequencing, Fluorescence

    Presynaptic inhibition is slower than ER Ca 2+ depletion following SERCA block (A-C) Average axonal ER Ca 2+ dynamics were measured using ER-GCaMP6-150 at 26°C, (A, blue) or 37°C (B, red). Grey traces are individual experiments, although some individual responses overshot the scale used here and their peaks were not included in the graph. Neurons were stimulated with 20 AP (20 Hz) and then treated with CPA to induce ER Ca 2+ depletion. After 3 min of CPA treatment the responses to a second stimulus were abolished (indicated by second arrow, 20AP 20Hz). (C) Estimate of Δ[Ca 2+ ] ER assuming an average resting [Ca 2+ ] ER based on ionomycin responses; n(26°C)=8, n(37°C)=15; n.s. p=0.36. (D) Single-AP cytosolic Ca 2+ responses (Fluo-5F AM, normalized to average pre-CPA ΔF response) every 60s measured before (black traces) and after CPA treatment at 26°C (D, G, blue traces) or 37°C (E, H, red traces). (G, H) Comparison of the kinetics of presynaptic inhibition and axonal ER Ca 2+ depletion. Curves were fit to single exponential decays where possible and time constants (τ) were obtained for comparison. τ ER (26°C)= 47.5 ± 3.4 s, n=8; τ ER (37°C)= 26 ± 0.9 s, n=15; τ cyto Ca 2+ (37°C)= 148 ± 18 s, n=7.

    Journal: Neuron

    Article Title: Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals

    doi: 10.1016/j.neuron.2017.01.010

    Figure Lengend Snippet: Presynaptic inhibition is slower than ER Ca 2+ depletion following SERCA block (A-C) Average axonal ER Ca 2+ dynamics were measured using ER-GCaMP6-150 at 26°C, (A, blue) or 37°C (B, red). Grey traces are individual experiments, although some individual responses overshot the scale used here and their peaks were not included in the graph. Neurons were stimulated with 20 AP (20 Hz) and then treated with CPA to induce ER Ca 2+ depletion. After 3 min of CPA treatment the responses to a second stimulus were abolished (indicated by second arrow, 20AP 20Hz). (C) Estimate of Δ[Ca 2+ ] ER assuming an average resting [Ca 2+ ] ER based on ionomycin responses; n(26°C)=8, n(37°C)=15; n.s. p=0.36. (D) Single-AP cytosolic Ca 2+ responses (Fluo-5F AM, normalized to average pre-CPA ΔF response) every 60s measured before (black traces) and after CPA treatment at 26°C (D, G, blue traces) or 37°C (E, H, red traces). (G, H) Comparison of the kinetics of presynaptic inhibition and axonal ER Ca 2+ depletion. Curves were fit to single exponential decays where possible and time constants (τ) were obtained for comparison. τ ER (26°C)= 47.5 ± 3.4 s, n=8; τ ER (37°C)= 26 ± 0.9 s, n=15; τ cyto Ca 2+ (37°C)= 148 ± 18 s, n=7.

    Article Snippet: All chemicals were obtained from Sigma except for cyclopiazonic acid (CPA (Alomone), thapsigargin and 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, Tocris), Fluo-5F, AM (Invitrogen), ionomycin (Alomone) and VGCC blockers isradipine, ω-conotoxin-GVIA and ω-Agatoxin IVA (Alomone).

    Techniques: Inhibition, Blocking Assay