Collagen 1 cellware six well plates

The following reagents/materials were used during cell culture: GO powder was purchased from TCI Chemicals, USA; Penicillinum crystalicum was purchased from Polfa Tarchomin, Poland; and Dulbecco’s Modified Eagle’s medium (DMEM) (1×) with Glutamax, fetal bovine serum (FBS), horse serum (HS), and antibiotics (AB), penicillin-streptomycin and amphotericin B, were purchased from Gibco, Life Technologies, USA. Phosphate-buffered saline (PBS), protease from Streptomyces griseus, and DMSO were purchased from Sigma-Aldrich, USA. Primaria tissue culture flasks (25 and 75 cm²) and Collagen I Cellware six-well plates were purchased from Becton Dickinson, USA.

The following materials were used during cell culture: the Ca salt (monohydrate) of HMB
(Ca-HMB) was purchased from Metabolic Technologies; Dulbecco’s Modified Eagle Medium
(DMEM) (1×) with glutamax, fetal bovine serum (FBS), horse serum (HS) and antibiotics
(AB) – penicillin–streptomycin and fungizone – were purchased from Gibco,
Life Technologies; penicillium crystalicum (AB) was purchased from Polfa Tarchomin; PBS,
protease from Streptomyces griseus and DMSO were purchased from Sigma
Aldrich. Tissue culture flasks Primaria (25, 75 cm²) and Collagen I Cellware
six-well plates were purchased from Becton Dickinson. Ca-HMB was transformed to the acid
form by acidification with 1 N-HCl. HMB was then extracted four times with diethyl
ether. The pooled organic layer was dried under vacuum for 24 h at 38 °C. The resulting
free acid was 99 % HMB as assessed by HPLC.