Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. musculus) |
BMP2-transfected C2C12 myoblast cells (C2C12-BMP2; C2-OB) |
Dr. M. Murshed, McGill University |
osteoblast cell line | |
| Strain, strain background (M.musculus, female) |
10 week-old C57Bl/6J | Jackson Laboratory | Stock #: 000664; RRID: IMSR_JAX:000644 |
primary osteoblast source, in vivo studies |
| Software, algorithm |
Calcium Analyser |
DOI: 10.3389/ fphys.2016.00525 |
MATLAB algorithm for calcium analysis |
|
| Chemical compound, drug | MANT-ATP | AnaSpec | Cat. AS-64610 | fluorescent ATP analog, 5 µM (overnight, 37°C) |
| Chemical compound, drug | GsMTx-4; GsM | Alomone Labs | Cat. STG-100 | Piezo1 inhibitor, 1 µM (10 min, room temperature) |
| Chemical compound, drug | U73122; U73 | Calbiochem | Cat. 662035 | PLC inhibitor, 10 µM (10 min, room temperature) |
| Antibody | GAPDH (D16H11, Rabbit antibody) |
Cell Signaling Technology |
Cat. 5174; RRID:AB_10622025 | WB, 1:1000 dilution, 5% BSA/TBST (1 hr, room temperature) |
| Antibody | β-tubulin (9F3, Rabbit antibody) |
Cell Signaling Technology |
Cat. 2128; RRID:AB_823664 | WB, 1:1000 dilution, 5% BSA/TBST (1 hr, room temperature) |
| Antibody | p-PKC (pan; βII Ser660, Rabbit antibody) |
Cell Signaling Technology |
Cat. 9371; RRID:AB_330848 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | PKCα (Rabbit antibody) |
Cell Signaling Technology |
Cat. 2056; RRID:AB_2284227 | WB, 1:1000 dilution,5% BSA/TBST (over night, 4°C) |
| Antibody | p-PKCδ/θ (Ser643/676, Rabbit antibody) |
Cell Signaling Technology |
Cat. 9376; RRID:AB_330848 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | PKCδ (D10E2, Rabbit anitbody) |
Cell Signaling Technology |
Cat. 9616; RRID:AB_10949973 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | p-PKD/PKCµ (Ser744/748, Rabbit antibody) |
Cell Signaling Technology |
Cat. 2054; RRID:AB_330848 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | PKD/PKCµ (D4J1N, Rabbit antibody) |
Cell Signaling Technology |
Cat. 90039 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | p-PKCζ/λ (Thr410/403, Rabbit antibody) |
Cell Signaling Technology |
Cat. 9378; RRID:AB_330848 | WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | PKCζ (C24E6, Rabbit antibody) |
Cell Signaling Technology |
Cat. 9368; RRID:AB_10693777 |
WB, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Antibody | anti-rabbit IgG, HRP-linked secondary antibody) |
Cell Signaling Technology |
Cat. 7074; RRID:AB_2099233 |
WB, 1:1000 dilution, 5% BSA/TBST (1 hr, room temperature) |
| Chemical compound, drug | Minimum Essential Medium (MEM) α; αMEM |
Gibco | Cat. 12,000–022 | cell culture reagent |
| Chemical compound, drug | Trypan Blue Solution, 0.4%; TB |
Gibco | Cat. 15250061 |
in vitro injury assay dye |
| Commercial assay or kit | Alamar Blue Cell Viability Reagent |
Invitrogen | Cat. DAL1025 | cell viability assay |
| Chemical compound, drug | DAPI | Invitrogen | Cat. D1306; RRID:AB_2629482 |
IF, nucleur stain, 1:5000 dilution, H20 (5 min, room temperature) |
| Chemical compound, drug | Fura2 AM | Invitrogen | Cat. F1221 | ratiometric calcium-binding dye (30 min, room temperature) |
| Chemical compound, drug | D-Luciferin potassium salt |
Invitrogen | Cat. L2916 | luciferase substrate, ATP bioluminescence assay |
| Chemical compound, drug | Lucifer Yellow CH, Lithium Salt; LY |
Invitrogen | Cat. L453 | GAP junction permeable dye, 10 µM (2 min, 37°C) |
| Chemical compound, drug | Rhodamine B-conjugated 10 000 MW Dextran; R-dextran |
Invitrogen | Cat. D1824 | in vitro injury assay dye |
| Chemical compound, drug | lysine-fixable Texas Red-conjugated 10 000 MW Dextra; LFTR-Dex |
Invitrogen | Cat. D1863 | in vivo injury assay dye |
| Antibody | Sclerostin (Goat antibody) | R and D Systems | Cat. AF1589; RRID:AB_2195345 |
IF, 1:1000 dilution, 5% BSA/TBST (over night, 4°C) |
| Chemical compound, drug | Collagenase P from Clostridium histolyticum |
Roche | Cat. 11213857001 | enzymatic digest of bone fragments |
| Commercial assay or kit | Cytotoxicity Detection KitPLUS; LDH |
Roche | Cat. 04 744 926 011 | LDH leakage assay |
| Antibody | Donkey anti-goat IgG-FITC |
Santa Cruz | Cat. Sc-2024; RRID:AB_631727 |
IF, 1:1000 dilution,5% BSA/TBST (1 hr, room temperature) |
| Chemical compound, drug | Adenosine 5’-triphosphate magnesium salt; ATP |
Sigma-Aldrich | Cat. A9187 | |
| Chemical compound, drug | Bisindolylmaleimide II; Bis | Sigma-Aldrich | Cat. B3056 | PKC inhibitor, 1 µM (10 min, room temperature) |
| Chemical compound, drug | Carbenoxolone disodium salt; Cbx |
Sigma-Aldrich | Cat. C4790 | Hemichannel blocker, 10 µM (10 min, room temperature) |
| Chemical compound, drug | Fast Red Violet LB Salt | Sigma-Aldrich | Cat. F3381 | alkaline phosphatase assay reagent |
| Chemical compound, drug | Gadolinium (III) chloride; Gd3+ |
Sigma-Aldrich | Cat. 439770 | Mechanically-sensitive channel blocker, 10 µM (10 min, room temperature) |
| Chemical compound, drug | GSK2193874; GSK | Sigma-Aldrich | Cat. SML0942 | TRPV4 inhibitor, 100 nM (10 min, room temperature) |
| Chemical compound, drug | HC-067047; HC | Sigma-Aldrich | Cat. SML0143 | TRPV4 inhibitor,100 nM (10 min, room temperature) |
| Chemical compound, drug | L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate |
Sigma-Aldrich | Cat. A8960 | osteoblast differentiation reagent,50 µg/mL |
| Chemical compound, drug | Luciferase from Photinus pyralis |
Sigma-Aldrich | Cat. L9420 | ATP bioluminescence assay reagent |
| Chemical compound, drug | ML218; ML | Sigma-Aldrich | Cat. SML0385 |
T-type VSCC inhibitor, 10 µM (10 min, room temperature) |
| Chemical compound, drug | Naphthol AS-MX phosphate disodium salt |
Sigma-Aldrich | Cat. N5000 | alkaline phosphatase assay reagent |
| Chemical compound, drug | N-ethylmaleimide; NEM | Sigma-Aldrich | Cat. E3876 | Vesicular activator, 1 mM (10 min, room temperature) |
| Chemical compound, drug | Nifedipine; Nif | Sigma-Aldrich | Cat. N7634 | L-type VSCC inhibitor, 10 µM (10 min, room temperature) |
| Chemical compound, drug | 1-octanol; Oct | Sigma-Aldrich | Cat. 297877 | Connexin blocker, 1 mM (10 min, room temperature) |
| Chemical compound, drug |
phorbol 12-myristate 13-acetate; PMA |
Sigma-Aldrich | Cat. P8139 | PKC activator, 100 nM (10 min, room temperature) |
| Chemical compound, drug | Quinacrine dihydrochloride |
Sigma-Aldrich | Cat. Q3251 | Vesicle dye, 10 µM (15 min, room temperature) |
| Chemical compound, drug |
Flufenamic Acid; FFA |
Tocris Bioscience | Cat. 4522 | Hemichannel blocker, 10 µM (10 min, room temperature) |
| Chemical compound, drug | Ionomycin calcium salt; Iono |
Tocris Bioscience | Cat. 1704 | calcium ionophore, 100 µM |
| Chemical compound, drug | PPADS tetrasodium salt; PPADS |
Tocris Bioscience | Cat. 0625 | P2R inhibitor,100 µM (10 min, room temperature) |
| Chemical compound, drug | Suramin hexasodium salt; Sur |
Tocris Bioscience | Cat. 1472 | P2R inhibitor, 100 µM (10 min, room temperature) |
| Chemical compound, drug | A 740003; A7 | Tocris Bioscience | Cat. 3701 | P2X7 inhibitor, 1 µM (10 min, room temperature) |
| Chemical compound, drug | Dulbecco’s Modified Eagle Medium; DMEM |
Wisent Bio Products | Cat. 319–020 CL | cell culture reagent |
| Chemical compound, drug | Fetal bovine Serum; FBS |
Wisent Bio Products | Cat. 080152 | cell culture reagent |
| Chemical compound, drug | Penicillin Streptomycin |
Wisent Bio Products |
Cat. 450–201-EL | antibiotic, 1% |
| Chemical compound, drug | Sodium Pyruvate | Wisent Bio Products |
Cat. 600–110 UL | cell culture reagent |
| Chemical compound, drug | Collagenase Type II | Worthington Biochemical Corporation |
Cat. LS004176 | enzymatic digest of bone fragments |