Cells were spheroplasted by gentle shaking at 30 °C for up to 45 min, washed with ice cold 1.2 M sorbitol and resuspended in 800 μl NP-S buffer (1.2 M sorbitol, 10 mM CaCl2, 100 mM NaCl, 1 mM EDTA (pH 8.0), 14 mM β-mercaptoethanol, 50 mM Tris-HCl (pH 8.0), 0.075% NP-40, 5 mM spermidine, 0.1 mM PMSF, 1% Sigma protease inhibitor cocktail (Sigma P8215)).
These solvents were further dried by gentle shaking with 3 Å molecular sieves (E. Merck, Mumbai, India).
Cells were dissociated for 5 hours by gentle shaking.
The dissociation of tissue was completed in KB medium by gentle shaking for 20 min and resting for 40 min at room temperature.
For the AS02 formulation, the contents of one vial of lyophilized PfAMA1 containing 62.5 µg of antigen (lot C) was mixed by gentle shaking with AS02 (approximately 0.6 ml) [32].
After hybridization, the slides were immediately placed in wash solution 1 (1×SSC and 0.1% SDS) to remove the cover slip and washed by gentle shaking in solution 1, 2 times for 5 min each; then washed in solution 2 (0.1×SSC and 0.1% SDS), 2 times for 10 min each; and finally in solution 3 (0.1×SSC), 4 times for 1 min each.
The washed 4-week-old leaves were briefly dried and cut into small leaf strips (0.5 mm) with a razor blade; then the strips were incubated in an enzyme solution [3% (w/v) cellulose R-10 (Yakult Honsha, Tokyo, Japan), 1.5% Macerozyme R-10 (Yakult Honsha), 0.4 M mannitol, 20 mM MES, 10 mM CaCl2, 0.2 mM KH2PO4, 1 mM KNO3, 1 mM MgSO4, 0.1 µM CuSO4, 10 µM KI, pH 5.8] in the dark by gentle shaking.
Excess stain was removed by washing with water and excess water was removed by gentle shaking.
The slices were deparaffinized in xylol exchange medium XEM-200 (Vogel, Giessen, Germany) by gentle shaking for 20 min.
In particular, microglial cells were obtained by mixed cultures of cortical glial cells (at in vitro day 14), by gentle shaking.
Each extraction was performed by gentle shaking for 18 h at 37 °C, with centrifugation at 27 000 g for 10 min between extractions.
