After perfusion, lungs were collected and processed by gentle mechanical disruption with forceps followed by enzymatic digestion with 2.4 U/mL dispase gradeII (Roche), 0.25 U/mL liberase TM Research Grade medium thermolysin (Roche), 20 Kunitz units/mL DNase (Sigma), and 20 U/mL hyaluronidase (Sigma), all dissolved in PBS.
These cells were infected with C. pneumoniae and lysed by mechanical disruption with a rubber policeman 72 hour post infection.
The hyphal mat was harvested, sequentially washed with PBS, and lysed by mechanical disruption with glass beads in a BeadBeater homogenizer (BioSpec Products, OK).
For thymocyte analysis, thymocytes were extracted by mechanical disruption with the base of a 5 ml syringe followed by filtration through 70 µm nylon mesh.
Finally, the lysate was subjected to mechanical disruption with a bead beater (BioSpec Products, Inc., Bartlesville, OK), on the Homogenize setting for 3 minutes.
MA-10 cells were resuspended in MSHE buffer (219 mM D-mannitol, 70 mM sucrose, 0.02% EGTA, 0,1% BSA, 1.8 mM Hepes pH 7.4) and subjected to mechanical disruption with 60 strokes with an insulin syringe.
After collection, all samples are processed with the QIAGEN RNeasy Kit to extract the RNA (mechanical disruption protocol with on-column DNase digestion).
Cells were pelleted by centrifugation, washed with Molecular biology grade water (Sigma, Taufkirchen, Germany) and lysed with a combination of Lysozyme/mutanolysin treatment and mechanical disruption of cells with glass beads [ 5].
Six weeks later, bacterial burden in lungs and spleen was assessed by mechanical disruption in PBS with 0.05% v/v Tween 80 and enumerated by colony assay.
To transition to a true high throughput (HTP) process, we also sought a means of removing the aggregates thus formed from the plates with minimal mechanical disruption, in a manner compatible with automation.
For all tissue samples, RNA was extracted with an optimized protocol for liver samples using mechanical disruption of tissue samples with zirconium beads and subsequent silica-based RNA extraction, as described elsewhere [ 10].
