Homogenization of Resilient Tissues by Bead Beating
Many tissues like muscle, heart, kidney and lung can sometime be difficult to homogenize due to the presence of elastic connective tissue. If approached correctly, bead beating can effectively homogenize these resilient tissues, as well as bone, and result in a highly fluid homogenate with small particles sizes.
The most important aspect in effectively homogenizing resilient tissue by bead beating is to not overload the homogenization container with sample. Deep well plates, microfuge tubes, and vials can all be used, but sample size must be matched to the container as the grinding balls used in each format are limited in the mass of sample it can disrupt. Thus generally, microfuge tubes and deep well plates should be limited to 50 mg of tissue while 4 ml grinding vials can handle up to 200 mg. Up to 1 gm of tissue can be processed in 15 ml vials. Customized larger vials have even been used to homogenize the complete gastrointestinal tract of a rat for pharmacokinetic analysis.
Pourrut, Xavier, Marc Souris, Jonathan S Towner, Pierre E Rollin, Stuart T Nichol, Jean-Paul Gonzalez and Eric Leroy. 2009. Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus. BMC Infectious Diseases 2009, 9:159
Protocol for Bead Beating
Plates, tubes, and vials should be loaded with sample and no more than 40% full with buffer. Dry grinder (i.e., grinding without buffer) will not work with animal tissues. Seal deep well plates with push on strip caps. We recommend round wells with polypropylene caps that push into the wells. Square well plates providing hiding places for tissue and the square push on mats can often leak. Microfuge tubes should be screw cap and the caps should have an "O" ring. Vials, whether polyethylene or polycarbonate, should have caps with liners. Additional, full racks of 4 ml tubes in the vial set configuration can be sealed with a press on mat.
Lock the plates, tubes, or vials into the homogenizer. Set for 2 minutes and run at 3/4 speed.
Remove the vessels and check the fluidity of the sample with a micropipette. If the tip clogs or pieces of tissue are still visible, process the sample for an additional 2 minutes and recheck.
Optimization of the sample can also be done by measuring the release of cellular enzymes, such as lactate dehydrogenase. To optimize, make multiple tubes of the same sample and process for different times, such as 2, 4, 6, 8, and 10 minutes. Centrifuge the samples to pellet debris and then perform an LDH assay (see link for assay details). The first time point with the highest LDH activity is the optimal processing time.