The resultant suspension was centrifuged at a low speed (3000 rpm, 5 min) to precipitate free curcumin.
The nanoparticles were washed with PBS and then resuspended in 1 ml of 0.05% BSA for 30 min to block free carboxylates, generating SA-FSNPs.
An effect of applied microwave frequency was studied at 300 °C for 3 min to produce free radicals (Additional file 1: Figure S2).
For the differentiation protocol, NIH-3T3 cells were grown to confluency in DMEM 10% FCS, they were fixed with PBS 3% formaldehyde (Sigma-Aldrich, France) for 15 min, washed with PBS and incubated with glycine (1 mM) (ICN Pharmaceuticals, France) for 15 min to saturate free formaldehyde sites.
Briefly, after incubation as described above, sperm suspensions were loaded with a fluorescent calcium probe Fluo-3 AM (5 µM final concentration; Invitrogen, Carlsbad, CA, USA) at 37°C for 30 min and then washed twice at 400 g for 5 min to remove free fluo-3 AM.
Briefly, cells were treated with 5 Gy of IR, and then fixed in PBS containing 4% paraformaldehyde (EMD Chemicals, Merck Corporation, San Diego, CA) at room temperature for 20 min. Fixed cells were rinsed with PBS and with 25 mM NH4Cl in PBS for 10 min to quench free aldehyde groups.
The membrane fraction was washed repeatedly by centrifugation at 21 000 × g for 10 min to remove free hemoglobin.
Samples were then incubated with iodoacetic acid (IAA, 50 mM) for 30 min to modify free cysteine residues.
The aqueous phase containing the SAG fraction was acidified with HCl to pH 1.0 and boiled for 30 min to separate free SA from conjugated SA.
After rinsing in PHEM the seedlings were incubated in 0.1% (w/v) NaBH4 in PHEM for 15 min to reduce free aldehydes.
