E6758

DNA-PAINT imaging was performed in 5× Buffer C (2.5 M NaCl; S7653, Sigma in 5x PBS; 14200-059, Gibco) supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA; E6758, Sigma), 2.5 mM 3,4-dihydroxybenzoic acid (PCA; 03930590, Sigma), 10 nM protocatechuate 3,4-dioxygenase pseudomonas (PCD; P8279, Sigma), and 1 mM ( ± )-6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid (Trolox; 238813-5G, Sigma). P strands (P1 and P5) were imaged at an imager strand concentration of 0.5 nM and acquisition rate of 150 ms, and R strands (R1 and R4) at a concentration of 50 pM and acquisition rate of 100 ms. All images were acquired with 50 EM gain, for 10,000 to 20,000 frames. Exchange PAINT was performed manually by adding the imaging buffer to the sample chamber and acquiring camera images. The buffer was then removed and the sample washed five times with 1× PBS to remove all imager strands. The subsequent imaging buffer containing another imager strand was then added and the procedure repeated until all targets were imaged.

For osteocalcin (OCN) detection, the ECM of the cells grown on 6-well plates was dissolved in 100 μl of EDTA (E6758, Sigma, 0.5 mol/L, pH 6.9). OCN content of the EDTA-solubilized ECM samples was quantified by an enzyme-linked immunosorbent assay (ELISA) (DY1419-05, DuoSet ELISA, R&D, Minneapolis, MN, United States) according to the manufacturer’s protocol.

The extraembryonic tissues (E7.5 and E8.5) or placentae (E9.5–E14.5) collected from one pregnant mouse were pooled together, completely cut into tiny pieces and rinsed 3 times with DMEM/F-12 1:1 cell culture medium (11320082, Gibco). The extraembryonic tissue (E7.5 and E8.5) pieces were digested with TrypLE™ Express Enzyme (1×, 12605010, Gibco) for 20 min at 37 °C. The placental tissue pieces were digested with collagenase Type IV (0.5 mg/mL, C5138, Sigma‒Aldrich) and DNase I (0.2 mg/mL, DN25, Sigma‒Aldrich) for 20 min at 37 °C. The released extraembryonic cells (E7.5 and E8.5) and placental cells were filtered through 40 µm cell strainers (352340, Falcon). To exclude the remaining red blood cells, the filtered cells were lysed with red blood cell lysis buffer [155 mM NH₄Cl (A9434, Sigma‒Aldrich), 10 mM KHCO₃ (237205, Sigma‒Aldrich), 0.1 mM EDTA (E6758, Sigma‒Aldrich)] for 3 min. To exclude cell debris and adherent cells, the released placental cells were centrifuged at 1200× g for 15 min after loading these cells onto a Percoll gradient consisting of 28% and 60% Percoll (17-0891-01, GE Healthcare), and only cells that were sedimented between 28% and 60% Percoll were collected. All purified single cells were suspended in DMEM/F12 cell culture medium for the following procedures.

For OCN detection, the ECM of the cells grown on 6-well plates was dissolved in 100 μL of EDTA (E6758, Sigma, 0.5 mol/L, pH 6.9). OCN content of the EDTA-solubilized ECM samples was quantified by an enzyme-linked immunosorbent assay (ELISA) (DY1419-05, DuoSet ELISA, R&D, Minneapolis, MN, USA) according to the manufacturer’s protocol. The observer who performed all the ELISA measurements was blinded to the group assignment.