Biopsy punch

Calvariae of miR21^fl/fl mice were harvested at 5 days of age, and two 5‐mm pieces of the bone were removed using a biopsy punch (Integra LifeSciences Corporation, Plainsboro, NJ, USA). Samples were washed in PBS and incubated in a 96‐well plate with α‐minimal essential medium supplemented with 10% FBS and 1% penicillin/streptomycin for 6 h. Bones were then treated with 2.5 μL per well of control adenovirus (Adeno‐GFP, cat.#1060) or Cre‐recombinase virus (cat.#1700) (Vector BioLabs, Malvern, PA, USA) diluted in serum‐free α‐minimal essential medium in a final volume of 45 μL overnight. Next, media were removed and samples were washed twice with PBS before adding α‐minimal essential medium supplemented with 10% FBS and 1% penicillin/streptomycin and cultured for additional 48 h at 37% and 5% CO₂. mRNA was quantified by qPCR and protein quantified by Western blotting.

In parallel with freezing down stimulated plasma, 40 μl was dropped onto two premarked 1.2 cm diameter circles on separate Protein Saver 903 cards (Whatman, Maidstone, UK) and dried for 3–4 h at ambient temperature (AT) in a horizontal position on the bench to produce dried plasma spots (DPS). The cards were then placed in zip-lock plastic bags (Whatman, Maidstone, UK) containing a desiccant and maintained at 4°C up to 8 months.
After this period, a biopsy punch (Integra Miltex, NY, USA) was used to cut out two discs from the cards of each patient. The discs were placed in the 96-well plate and 70 μl of the elution buffer (2% BSA, 0.1% Tween-20 in PBS 1X) was added. The plate was incubated at ambient temperature for 1 hour in a shaker to elute all cytokines and chemokines of plasma deposited on filter paper.