Modulation of the [Ca2+]i was performed on washed RBCs in suspension at room temperature (RT) under continuous agitation in (i) DMEM containing 0.3 µM of the Ca2+ ionophore A23187 (Sigma-Aldrich) for 10 min; (ii) A23187-containing DMEM for 10 min followed by reincubation in A23187-free medium for another 10 min; or (iii) Ca2+-free homemade medium containing 1 mM Ca2+-chelating agent ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA, Sigma-Aldrich) for 10 min. All RBCs were then pelleted at 133 g for 2 min, resuspended in the adequate medium and either used in suspension for [Ca2+]i measurement or hemoglobin release or labelled, spread onto PLL-coated coverslips and observed by vital microscopy for lipid domains (see below) or Ca2+ determination. To evaluate [Ca2+]i, washed RBCs were preincubated in suspension at RT with 5 µM Fluo-4 (Invitrogen) in Ca2+-free medium for 60 min under continuous agitation, pelleted at 133 g for 2 min and resuspended in Ca2+-free medium, then treated with the above modulators and either measured for Ca2+ in 96-well plates (excitation 490 nm, emission 520 nm; SpectraCountTM, Packard BioScience Co.) or spread onto PLL-coated coverslips for intracellular Ca2+ signal (see below).
A23187
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Japan, Sao Tome and Principe, China
A23187 is a calcium ionophore, a chemical compound that facilitates the movement of calcium ions (Ca2+) across cell membranes. It is commonly used as a research tool in cell biology and biochemistry studies.
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Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (23 mentions)
Thapsigargin, by Merck Group (13 mentions)
Ionomycin, by Merck Group (10 mentions)
Sytox green, by Thermo Fisher Scientific (10 mentions)
Acetylcholine, by Merck Group (9 mentions)
Fluo 4 am, by Thermo Fisher Scientific (8 mentions)
Adenosine, by Merck Group (6 mentions)
Cycloheximide (chx), by Merck Group (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Tunicamycin, by Merck Group (5 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (5 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (5 mentions)
Bapta am, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Propidium iodide, by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Bx53 microscope, by Olympus (5 mentions)
Fitc psa, by Merck Group (4 mentions)
Adenosine triphosphate (atp), by Merck Group (4 mentions)
Thrombin, by Merck Group (4 mentions)
280 protocols using a23187
1
Modulating Intracellular Calcium in RBCs
2
Alginetin-induced Cellular Parameter Changes
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Alginetin (3–100 mM in 2 μL of DMSO) was added to cell suspensions (1.998 mL per one test tube) and the mixtures were incubated at 36–37°C. Each cell suspension (100 μL) was analyzed using flow cytometry to assess alginetin-induced changes in cellular parameters. Fluorescence data acquisition from 3 × 103 cells required 10–15 s. In our previous study to examine the cytotoxicity of H2O2 as oxidative stress and A23187 (Calcimycin), a divalent cation ionophore (Sigma-Aldrich Co, St. Louis, MO, USA) as Ca2+ overload [6 (link)], the cells were incubated with H2O2 or A23187 for 3–4 h to induce cell death in 20–40% of cells. Change in [NPT]i by alginetin was examined 2 h after the application in order to suggest the [NPT]i before the occurrence of cell death induced by H2O2.
3
Calcium-Induced α-Synuclein Phosphorylation
4
Sperm Acrosome Reaction Analysis
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The A23187-induced acrosome reaction was analyzed as previously reported.38 (link) Briefly, sperm were collected from the upper portion of HTF medium, and the calcium ionophore A23187 (Sigma) was added (final concentration 10 μM) to induce the acrosome reaction. Fifteen minutes later, the sperm were spotted on a glass microscope slide, dried at room temperature and fixed with methanol for 30 s. Intact acrosomes were stained with FITC-PNA, and the sperm nuclei were labeled with DAPI. Sperm that had undergone the acrosome reaction could not be labeled with FITC-PNA. More than 200 sperm were examined for all experimental conditions.
5
Monitoring HEK293T Cell Calcium Signaling
6
Intracellular Ca2+ Dynamics in Osteoclasts
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Osteoclasts derived from Ano1fl/fl, Ctsk-Cre;Ano1fl/fl, WT, and Ano1 TG mice were seeded on confocal dish and induced by RANKL for 5 days to measure intracellular Ca2+51 (link). The cells were loaded with 5 μM fluo-4, AM (Molecular Probe) for 20 min at 37 °C in Tyrode solution, then rinsed twice with Tyrode solution and mounted on the inverted stage of a confocal scope. Fluorescence excitation was performed using 488 nm laser, and detection filters were set at 530 nm. Images were acquired every 3 s and analyzed using Interactive Data Language (IDL, Research Systems) software. Cells were scanned for 20–30 s to obtain Fresting (F), then replaced the solution with 0 Ca2+ Tyrode solution including 4 mM EGTA (Invitrogen), 5 μM thapsigargin (Molecular probes), and 10 μM A23187 (Sigma). Stored calcium was released to the cytoplasm immediately. We defined the peak value as FER release. Added 100 μM BAPTA, AM into solution to obtain Fmin. Then replaced the solution with 10 mM Ca2+, 5 μM thapsigargin, 12 μM A23187 (Sigma), 3 μM FCCP (Sigma), and 20 mM 2-DG (Sigma) in Tyrode solution. The stable value was Fmax. Finally, [Ca2+]i was calibrated using the equation [Ca2+] = Kd×(F–Fmin)/(Fmax–F).
7
Murine Mast Cell Activation and Histamine Quantification
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The murine mast cell line MC/9 was maintained in DMEM media containing 10% (v/v) FBS, 0.05 mM 2-mercaptoethanol (Sigma Chemical Co., MO, U.S.A.), 10% (v/v) Rat T-STIM (BD Biosciences, MA, U.S.A.), 100 U/mL of penicillin and 100 μg/mL of streptomycin in a humidified 5% CO2 atmosphere. The MC/9 cells were plated in 48-well plates at a concentration of 2×105 cells per well. The cells were either untreated or treated with either phorbol 12-myristate 13-acetate (50 nM; PMA, Sigma-Aldrich, Inc., MO, U.S.A.) and A23187 (1 μM; Sigma-Aldrich, Inc., MO, U.S.A.) alone or PMA/A23187 (PA) + AC (50–200 μg/mL) for 24 hr. The histamine levels in the MC/9 cell supernatants were measured by ELISA in accordance with the manufacturer’s instructions (Oxford Biomedical Research, U.S.A.).
8
Sperm Capacitation Induction Protocols
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Two treatments were prepared for each of 10 semen samples. Cap and Non‐Cap treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr. For seven of these samples, a third treatment, Cap + ionophore, was prepared in which sperm were incubated with 2‐hydroxypropyl‐β‐cyclodextrin for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich, Allentown, PA; reference C7522) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
In a second set of experiments, Cap (n = 4) and Non‐Cap (n = 5) treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr, plus a third treatment of Non‐Cap + ionophore was prepared. For this treatment, sperm were incubated in basal non‐capacitating media for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
Following incubation, the sperm were attached to slides for 0.25 hr, labeled for 10 min with 10 μg/ml of Alexa Fluor® 647‐conjugated PNA from Arachis hypogaea (Thermo Fisher, Allentown, PA; reference L32460), washed 1× with mHTF, fixed for 0.5 hr, and then labeled with 2 μg/ml of Alexa Fluor 488‐conjugated CTB (Thermo Fisher, reference C34775). All labeling and slide work was done in a humidified chamber maintained at 37°C.
In a second set of experiments, Cap (n = 4) and Non‐Cap (n = 5) treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr, plus a third treatment of Non‐Cap + ionophore was prepared. For this treatment, sperm were incubated in basal non‐capacitating media for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
Following incubation, the sperm were attached to slides for 0.25 hr, labeled for 10 min with 10 μg/ml of Alexa Fluor® 647‐conjugated PNA from Arachis hypogaea (Thermo Fisher, Allentown, PA; reference L32460), washed 1× with mHTF, fixed for 0.5 hr, and then labeled with 2 μg/ml of Alexa Fluor 488‐conjugated CTB (Thermo Fisher, reference C34775). All labeling and slide work was done in a humidified chamber maintained at 37°C.
9
Sperm Capacitation and Acrosome Reaction
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Sperm were incubated with (Cap) or without (NonCap) CD. Day0‐A23187 treatment sperm were incubated with CD for 2.5 hr and then calcium ionophore A23187 (Sigma–Aldrich, St. Louis, MO; catalogue # C7522) was added to a final concentration of 20 μM for 0.5 hr prior to fixation and evaluation. Day1‐A23187 sperm were first incubated overnight with CD. The following day, A23187 was added to a final concentration of 20 μM and the cells were incubated for another 0.5 hr, fixed and evaluated.
A 6.36 mM solution of progesterone (P4; SigmaP8783) was prepared in pure ethanol (Sigma 459836), which was further diluted to create a 254.4 μM stock solution in mHTF. This P4 stock solution was stored at −20 °C until use. Day0‐P4 sperm were incubated with CD for 2.5 hr and then the P4 stock was added to yield a final concentration of 10 μM P4. Sperm were incubated in the presence of P4 for 0.5 hr prior to fixation and evaluation. Day1‐P4 sperm were incubated in the presence of CD overnight. The following day, P4 was added to a final concentration of 10 μM and the cells were incubated for another 0.5 hr, fixed, and then evaluated.
A 6.36 mM solution of progesterone (P4; SigmaP8783) was prepared in pure ethanol (Sigma 459836), which was further diluted to create a 254.4 μM stock solution in mHTF. This P4 stock solution was stored at −20 °C until use. Day0‐P4 sperm were incubated with CD for 2.5 hr and then the P4 stock was added to yield a final concentration of 10 μM P4. Sperm were incubated in the presence of P4 for 0.5 hr prior to fixation and evaluation. Day1‐P4 sperm were incubated in the presence of CD overnight. The following day, P4 was added to a final concentration of 10 μM and the cells were incubated for another 0.5 hr, fixed, and then evaluated.
10
Acrosome Reaction Analysis of Sperm
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The rate of acrosome reaction was analyzed as described previously (Nakanishi et al., 1999 (link)) The Fam71f1-mutant line was crossed with B6D2 Tg mice carrying CAG/Su9-DsRed2, Acr3-EGFP (Red Body Green Sperm; RBGS). Cauda epididymal spermatozoa were dispersed in a drop of TYH medium. After 10 min or 4 h incubation at 37°C under 5% CO2, 10 µg/ml of PI was added to the drop and, subsequently, an aliquot of the sperm suspension was placed on a glass slide; the acrosome reaction was determined by observing EGFP signals while distinguishing viable cells with PI staining with a BX-53 microscope (Olympus). To induce the acrosome reaction with Ca2+ ionophore A23187, 20 μM A23187 (100106, MERCK) was added after 4 h incubation. After 10 min of incubation, PI was also added and the acrosome reaction was analyzed as described above.
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