Sample incubation conditions were based on literature (9, 11, 12): 20 min at 37 [degrees]C for calcein
AM and calcein
violet, 60 min at 37 [degrees]C for CFSE, and 60 min at room temperature for di-8-ANEPPS and lactadherin.
Viability analysis was per formed on the same pictures using combined information from H-33342 and calcein
Amplification can be detected through visualization with naked eye, due to the formation of a white precipitate of magnesium pyrophosphate or the change of color of the solution by using dyes (SYBR Green, calcein
, HNB, picogreen).
Cytotoxicity studies using FACS (Figure 2S) showed no difference in viability measured by calcein
staining, demonstrating that the membrane integrity of the cells was not compromised and that the MSCs remained highly viable after 24 hours of exposure to PAA-GNTs.
Cell viability was verified in the agarose construct using calcein
AM and propidium iodide fluorescent (live-dead) staining (Figure 3(a)).
The cells were gently washed with serum-free medium, and calcein
AM-labeled THP-1 cells (5 x [10.sup.4]/ml DMEM medium) (Sigma, USA) were then added to the endothelial cells.
Tetracycline (12 mg/Kg body weight) on 7, 14, 21st days; Alizarin red S (30 mg/Kg body weight) on 28, 35 and 42nd days; Calcein
(5 mg/Kg body weight) on 48, 56 and 63rd days.
With the purpose of material cytotoxicity assays performed with calcein
and ethidium homodimer (Thermo[R]), different dilutions of silver nanoparticles synthesized by a green method were made using an extract of Annona muricata.
The hematopoietic progenitor cells (HPCs) ([Lineage.sup.-]/[Sca-1.sup.-]/[c-kit.sup.+], [LSK.sup.- ]) and HSCs ([Lineage.sup.-]/[Sca-1.sup.+]/[c-kit.sup.+]; [LSK.sup.+]) were analyzed as previously described (14), and the levels of intracellular ROS and LIP were analyzed by measuring the MFI of 2'-7'dichlorofluorescein or calcein
using a flow cytometer.
For live cell staining, 2.5 [micro]M of calcein
AM (Biotium, Fremont, CA) was added in each well and incubated for 30 minutes before the fluorescent intensities (ex/em: 495 nm/517 nm) were recorded by the plate reader.
Following incubation with the reagents cells must be washed to reduce the possibility of background fluorescence from unspecific extracellular binding of ethidium homodimer or hydrolysis in aqueous solution of AM calcein
. The fluorescence of cells alone or cell suspension or tissue can be detected in the fluorescence microscope, fluorescence spectroscopy or microplate reader using appropriate filters.