Calcium ionophore a23187

Manufactured by Merck Group

Sourced in United States, Germany, Canada, France, Sao Tome and Principe, Spain

Calcium ionophore A23187 is a chemical compound that facilitates the movement of calcium ions (Ca2+) across cell membranes. It is commonly used as a research tool to study calcium-mediated cellular processes.

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136 protocols using calcium ionophore a23187

Comprehensive Sperm Analysis Workflow

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The cauda epididymides were dissected and then placed in pre-warmed (37°C) Tyrode’s buffer (Sigma-Aldrich) to allow dispersion of sperm. After 15 min, sperm suspensions were collected and sperm motility, progressive motility, and concentration were analyzed by CASA (Hamilton Thorne). Moreover, in sperm motility rescue assays, sperm suspensions were incubated with 10 μM db-cAMP (Sigma-Aldrich) or 0.5 μM calcium ionophore A23187 (Sigma-Aldrich) for 15 or 30 min, and then sperm motility was analyzed by CASA.
For teratozoospermia analysis, the sperm pellet was initially smeared on a glass slide. After reaching dryness at room temperature, the slide was fixed and stained as described in the Diff-Quick method (BRED Life Science Technology Inc.). The slide was viewed under a microscope (Olympus BX53). On the other hand, for electron microscopy analysis, the sperm pellet was fixed overnight in 2.5% glutaraldehyde buffer.
For sperm acrosome reaction analysis, sperm suspensions were incubated with 5 μM calcium ionophore A23187 for 30 min, and then FITC–peanut agglutinin (PNA) (Sigma-Aldrich) was applied to identify sperm acrosomal status and detected with laser scanning confocal microscope. The percentage of acrosome-reacted sperm was calculated by evaluating at least 300 sperm per test.

Liu Y., Zhang C., Wang S., Hu Y., Jing J., Ye L., Jing R, & Ding Z. (2020). Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression. Journal of Molecular Cell Biology, 12(7), 515-529.

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Measuring Sperm Acrosome Reaction

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The spontaneous and calcium ionophore-induced acrosome reactions (ARs) were measured before freezing and after thawing by using fluorescein isothiocyanate-labelled Pisum sativum agglutinin (Sigma-Aldrich). Viable spermatozoa with damaged acrosomes were interpreted as spontaneous acrosome-reacted spermatozoa, which were measured according to the method of Esteves et al. (12) . The induced AR was assessed following 2010 WHO guidelines. Briefly, an aliquot of washed spermatozoa was diluted to 3 Â 10 7 /mL in BWW and incubated for 3 hours at 37 C in an atmosphere of 5% (v/v) CO 2 in air to allow capacitation. A total of 10 mmol/L calcium ionophore A23187 (Sigma-Aldrich) was then added to the sperm suspension, followed by incubation for 15 minutes at 37 C to induce AR, and equal volume of dimethyl sulfoxide was added to another sperm suspension instead of the calcium ionophore A23187 as control. Subsequently, the samples were fixed on a glass slide with absolute methanol, air dried, and incubated for 1 hour with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (working solution, 25 mg/mL) at 4 C. After washing and mounting, at least 500 spermatozoa per slide were assessed by a fluorescence microscope, and the percentage of AR after ionophore challenge was presented as the test %AR minus the control %AR.

Liu J., Wang W., Liu X., Wang X., Wang J., Wang Y., Li N, & Wang X. (2018). Supplementation of cryopreservation medium with TAT-Peroxiredoxin 2 fusion protein improves human sperm quality and function. Fertility and sterility, 110(6).

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Evaluation of Bull Sperm Capacitation

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Frozen-thawed semen samples were purified in fertilization medium containing heparin (Sigma, USA) by the ‘swim-up’ method for 30 min. Capacitated sperms were collected and stimulated with 20 µM calcium ionophore A23187 (Sigma, USA) for 1 h. The processes of sperm capacitation and acrosome reaction were assessed using the chlortetracycline (CTC) fluorescence assay method according to previous report for bull sperm [49 (link)]. MMP (ΔΨm) was measured by using the fluorescent dye, JC-1 (Beyotime, China), as described previously [50 (link)]. Sperm images were acquired on the same day using Biotek Cytation5 (Biotek, USA) and an EVOS FL fluorescence microscope (Thermo, USA). Two hundred sperms were counted for each sample.

Luo X., Liang M., Huang S., Xue Q., Ren X., Li Y., Wang J., Shi D, & Li X. (2023). iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm. BMC Genomics, 24, 245.

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Evaluating Probiotic Effects on Intestinal Immunity

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C57BL/6 J mice were orally given 100 µl of PBS, B. longum 2012, or B. longum 420 (2 × 10⁹ CFUs/100 µl of PBS) every day. On day 29, animals (n = 8/group) were killed to remove the small intestine including the PPs, MLNs, and colon. The expressions of interferon (IFN)-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells in the PPs, MLNs, small intestine, and colon were analyzed with the Cytofix/Cytoperm™ Plus Kit with GolgiStop™ (BD Biosciences) according to the manufacturer’s instructions. In brief, the cells were incubated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich), 5 μM calcium ionophore A23187 (Sigma-Aldrich), and GolgiStop™ in RPMI 1640. Surface staining was conducted with the anti-CD8-APC-Cy7 antibody (53-6.7, BD Biosciences) and the anti-CD4-Percp-Cy5.5 antibody (CK1.5, BD Biosciences), and intracellular cytokine staining was conducted with the FITC-conjugated anti-IFN-γ antibody (XMG1.2, BD Biosciences) [16 (link)]. Samples were analyzed using the FACSCanto™ II flow cytometer and the FlowJo™ software.

Nakagawa N., Hashii Y., Kayama H., Okumura R., Nakajima H., Minagawa H., Morimoto S., Fujiki F., Nakata J., Shirakawa T., Katayama T., Takeda K., Tsuboi A, & Ozono K. (2022). An oral WT1 protein vaccine composed of WT1-anchored, genetically engineered Bifidobacterium longum allows for intestinal immunity in mice with acute myeloid leukemia. Cancer Immunology, Immunotherapy, 72(1), 39-53.

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CaMKII Signaling in Cellular Stress Response

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GAS (SMB00313), CoCl₂ (232696), and calcium ionophore (A23187) were purchased from Sigma-Aldrich (St Louis, MO, USA). GAS and CoCl₂ were dissolved in phosphate-buffered solution to prepare the original solution of 100 mM, which was stored at -20° C. CQ diphosphate (HY-17589) and 3-BDO (HY-U00434) were purchased from MedChemExpress. Fluo-4AM (S1060) was obtained from Beyotime Biotechnology, Inc. (Nanjing, China). Primary antibodies against β-actin (66009-1-Ig, 1:10 000), LC3 (14600-1-AP, 1:1000), p62 (18420-1-AP, 1:1000), p62(66184-1-lg), LAMP-2 (66301-1-lg, 1:1000), and CaMKIIα (11533-1-AP, 66843-1-Ig) were purchased from Proteintech (Wuhan, China). Rabbit anti-p-p62 (Thr349) (Ab211324) was purchased from Abcam (Cambridge, MA, USA). Primary antibodies against CaMKIIα (#4436, 1:1000) and anti-p-CaMKIIα (Thr286) (#12716, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit IgG (H&L)-HRP (BS13278, 1:10000) and goat anti-mouse IgG (H&L)-HRP (BS12478) were obtained from Bioworld. Goat PAb to Rb IgG Alexa Fluor®488 (Ab150077, 1:200) and goat PAb to MS IgG Alexa Fluor®647 (Ab150115) were purchased from Abcam. A fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (A0516, 1:200) was purchased from Beyotime Biotechnology.

Chen T.T., Zhou X., Xu Y.N., Li Y., Wu X.Y., Xiang Q., Fu L.Y., Hu X.X., Tao L, & Shen X.C. (2021). Gastrodin ameliorates learning and memory impairment in rats with vascular dementia by promoting autophagy flux via inhibition of the Ca2+/CaMKII signal pathway. Aging (Albany NY), 13(7), 9542-9565.

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Boar Sperm Acrosome Reaction Induction

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Fresh boar semen samples (breed: Tempo (Topigs Norsvin breeding line), AIM the Netherlands, Vught, The Netherlands)) were obtained at a concentration of 20 × 10⁶ cells·mL⁻¹ and stored at 17 °C. The control samples were diluted in 1x phosphate-buffered saline (PBS, Sigma-Aldrich, Buchs, Switzerland) to a concentration of 2 × 10⁶ cells·mL⁻¹. The sample was treated with calcium ionophore A23187 (Sigma-Aldrich, Buchs, Switzerland) to induce the acrosome reaction. Briefly, 300 µL of fresh boar sample was centrifuged and the pellet was resuspended in 300 µL Solusem Bio+ sperm diluent (AIM worldwide, Vught, The Netherlands) spiked with 3 mM Ca³⁺ (Sigma-Aldrich, Buchs, Switzerland). The ionophore was added to a final concentration of 50 µM and the sample was incubated for 30 min at 36 °C. After washing, the sample was resuspended in 1x PBS to a concentration of 2 × 10⁶ cells·mL⁻¹. The viability and acrosomal status of the control (untreated freshly diluted spermatozoa) and treated sample was evaluated by propidium iodide (PI, Life Technologies, Eugene, OR, USA) and peanut agglutinin antibodies conjugated to Alexa Fluor 488 (PNA-AF488, Invitrogen, Waltham, MA, USA).

Kruit S.A., de Bruijn D.S., Broekhuijse M.L., Olthuis W, & Segerink L.I. (2022). Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa. Biosensors, 12(9), 679.

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Quantifying Tachyzoite Egress in Toxoplasma gondii

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A total of 1 × 10⁵ tachyzoites were inoculated onto HFFs, grown in a 24-well plate, washed at 2 h postinfection, and then allowed to replicate in the absence or presence of ATc for 30 h. Intracellular parasites were then incubated with DMEM containing 0.15% dimethyl sulfoxide (DMSO) or 3 µM calcium ionophore A23187 (Sigma) in DMSO for 5 min at 37°C before fixation with 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Percentages of egressed vacuoles were assessed by IFA with an anti-GRA3 antibody (55 (link)) to visualize the extent of vacuole lysis and an anti-SAG1 antibody (54 (link)) to identify parasite spreading. Independent experiments were conducted three times, and 250 vacuoles were counted for each condition.

Lévêque M.F., Berry L., Cipriano M.J., Nguyen H.M., Striepen B, & Besteiro S. (2015). Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii. mBio, 6(6), e01446-15.

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Cytokine-Producing Cell Phenotyping in BAL

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The bronchial lavage (BAL) fluid samples were centrifuged (4°C, 10 min, 400 g). The resultant supernatant was frozen (−80 °C) and the cells were cryopreserved (−150 °C) in 90% FBS and 10% DMSO. We used the frozen supernatant and cryopreserved cells in our analysis (IRB approval #2015–7983). Characteristics of the subjects are provided in Table S3. After thawing, we incubated (4°C, 30 min) the cells (0.5 × 10⁵) with anti-human CD3 (UCHT1), anti-human CD5 (UCHT2), anti-human CD11c (S-HCL-3), anti-human CD11b (M1/70), anti-human CD19 (HIB19), anti-human FcεRI (AER-37), anti-human CD34 (581), anti-human CD14 (HCD14), anti-human CD45 (HI30), anti-human CD127 (A019D5) and isotype-matched control antibodies (MOPC-173) (all diluted 1:100). The cells were then treated with 10 ng/ml phorbol 12-myristate 13-acteate (PMA) and 500 ng/ml calcium ionophore A23187 (both from Sigma-Aldrich) (4 °C, 60 min). The cells were then washed and fixed (4 °C, 60 min) in 1X Cytofix/Cytoperm buffer (BD Biosciences) and permeabilized (4 °C, overnight) using 1X Permeabilization Buffer (BD Biosciences) according to manufacturer instructions. The permeabilized cells were incubated with anti-human GATA-3 (TWAJ), anti-human RORγT (AFKJS-9) and anti-human IL-22 antibody (22URTI) (all 1:100, all eBiosciences) as described before (De Grove et al., 2016 (link)).

Oherle K., Acker E., Bonfield M., Wang T., Gray J., Lang I., Bridges J., Lewkowich I., Xu Y., Ahlfeld S., Zacharias W., Alenghat T, & Deshmukh H. (2020). Insulin like growth factor 1 supports a pulmonary niche that promotes type 3 innate lymphoid cell development in newborn lungs.. Immunity, 52(2), 275-294.e9.

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Hepatocyte Prostaglandin E Metabolism

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Isolated hepatocytes were cultured in serum-free medium with/without arachidonic acid (AA) (Cayman,Ann Arbor, MI) (1μM) and/or 15-PGDH inhibitor (EMD Millipore, Billerica, MA) (1μM) for 6h. Before collecting culture medium, cells were stimulated with Calcium ionophore A23187 (Sigma,St. Louis, MO) (10μM) for 10min. Prostaglandin E Metabolite concentration in culture medium was analyzed according to the instruction of Prostaglandin E Metabolite EIA Kit (Cayman, Ann Arbor, Michigan).

Yao L., Chen W., Song K., Han C., Gandhi C.R., Lim K, & Wu T. (2017). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) prevents lipopolysaccharide (LPS)-induced acute liver injury. PLoS ONE, 12(4), e0176106.

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Tachyzoite Egress Regulation by Calcium Homeostasis

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Asexual reproduction of T. gondii culminates with the egress of the newly formed tachyzoites from the surrogate host cell and subsequent parasite invasion into new host cells. Therefore, parasite egress represents a very important event which is indispensable for the parasite dissemination in the host body. In order to determine the role of the GRAs in this important process, we examined the effect of ionophore A23187 modulating Ca2⁺ homeostasis on the egress of the parasite from the host cells. Briefly, HFF cells were incubated with 10⁵ freshly harvested T. gondii tachyzoites in 2 ml culture medium for 3 h, followed by washing twice with DMEM medium to remove the unbound parasites. After 30–36 h of incubation, the wells were washed twice with sterile PBS and 3 μM calcium ionophore A23187 (Sigma) diluted in DMSO were added to the HFF cells. Live cell microscopy was used to monitor the timing of parasite egress from HFF cells infected with the WT strain compared with HFF cells infected with the mutant strains after addition of 3 μM calcium ionophore A23187.

Bai M.J., Wang J.L., Elsheikha H.M., Liang Q.L., Chen K., Nie L.B, & Zhu X.Q. (2018). Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System. Frontiers in Cellular and Infection Microbiology, 8, 300.

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