Homogenization of Mouse Muscle: A Comparison of Methods
The disruption of biological samples is a necessity for assays aiming to quantify RNA, DNA, and many proteins. Both chemical and mechanical methods are available for sample disruption, with chemical methods being valuable for many common cells (e.g., E. coli and cultured cells). However, many microorganisms and intact tissue mass are not efficiently disrupted by chaotropes and detergents, thus mechanical methods relying on shearing and grinding forces must be employed. Mechanical homogenizers, manual homogenizers, mortar and pestles, sonicators, mixer mills, and vortexers are several of the more common tools used for mechanical disruption.
Different methods of mechanical homogenization lead to different forms of lysate. For instance, the glass Dounce homogenizer is often used to disrupt cultured cells for the preparation of intact nuclei and microsomes, a process dependent upon the clearance between the Dounce pestle and wall of the tube. Alternatively, sonication destroys cells and often dissipates cell organelles and subcellular components. As many tests require partial lysis of a sample, as with a presence/absence PCR assay, while other require complete homogenization (as with pharmacokinetic analysis), an evaluation of the effectiveness of homogenization methods is relevant.
Materials and Methods
Tissue Samples: Female CD1 mice, 42 days (Charles River Laboratories) were sacrificed and leg muscles were extracted, frozen, and stored at -140°C.
Lactate Dehydrogenase Assay: Mouse muscle was homogenized and lysates were assayed for the release of lactate dehydrogenase from cells. Several one-step methods and two-step homogenization methods were employed. For one-step methods, a known mass of muscle was placed in a tube and 10 volumes of Complete Homogenization Buffer with protease inhibitors was added (OPS Diagnostics), followed by homogenization as described in Table 1. For two-step homogenization methods, homogenization protocols were run in tandem (e.g., mechanical homogenization followed by sonication). For all approaches, total muscle lysates were cleared by centrifugation prior to analysis to remove LDH bound to debris and tissue particles. A colorimetric NAD linked assay was used to measure released lactate dehydrogenase. The LDH substrate solution is prepared by mixing 2 ml of 1 M TRIS, pH 8, with 8 ml water, followed by the addition of 49 mg lithium lactate (Sigma L-1500), 100 μl of INT stock (33 mg/ml iodonitrotetrazolium chloride (Sigma I-8377) in DMSO), 0.9 mg phenazine methosulfate (Sigma P-9625), and 8.6 mg β-nicotinamide adenine dinucleotide (SigmaN-0632). Briefly, 50 μl of lysate (or lysate diluted in PBS for concentrated samples) is added to a well of a microtiter plate followed by 125 μl of substrate solution. The plate is placed in a kinetic plate reader and optical densities at 490 nm are measured every 30 sec. for 5 min. LDH activity is calculated as the maximum slope of the reaction.
Homogenization: Homogenization was performed with a variety of conventional and high throughput devices. Table 1 summarizes the methods and tools used.
Table 1. Homogenization Tools and Methods
|Dounce Homogenizer (LabGlass, Buena, NJ)||Sample is placed in the tube, buffer is added, and a Type B pestle is pressed down onto the sample. With pressure, the pestle is turned 360°, and then slowly removed from the tube (a strong vacuum helps to create shearing forces). This action is repeated nine more times.|
|Potter-Elvehjem Homogenizer(LabGlass, Buena, NJ)||Sample is placed in the tube, buffer added, and then a Teflon pestle driven by Talboy Mixer (Troemner, Thorofare, NJ) at 650 rpm is pressed firmly down onto the sample for 5 sec. The pestle is slowly removed due to a vacuum. This action is repeated nine more times.|
|Conical Tissue Grinder(LabGlass, Buena, NJ)||Sample is placed in the tube, buffer is added, and a Type B pestle is pressed down onto the sample. With pressure, the pestle is turned 360°, and then slowly removed from the tube (a strong vacuum helps to create shearing forces). This action is repeated nine more times.|
|CryoGrinder (OPS Diagnostics, Lebanon, NJ)||This is a mechanically driven, cryogenic mortar & pestle system. Muscle is placed in porcelain mortar, chilled in liquid nitrogen, and then ground with zirconium/porcelain pestles powered by a handheld cordless screwdriver. Powdered tissue was handled in a CryoCooler (OPS Diagnostics, Bridgewater, NJ) to prevent thawing, and transferred to a chilled 15 ml screw capped tubes. For prolonged storage, powdered tissue was transferred to a -140°C freezer.|
|Mixer Mill (HT Homogenizer) (OPS Diagnostics, Lebanon, NJ)||This high throughput bead beater grinds samples using stainless steel balls. Samples were homogenized in either deep well plates (DWP) with a 5/32" ball or 15 ml vial sets (15 VS) with two 7/16" balls. All samples were homogenized for 2 min. at 1500 rpm.|
|Mechanical Homogenizer (Tempest) (VirTis, Gardiner, NY)||This traditional rotor/stator-type mechanical homogenizer was used with a 7 mm tip (generator). Each sample was processed for 1 min. at 10 rpm.|
|Sonicator (Virsonic 100)(VirTis, Gardiner, NY)||A set volume of 0.5 ml was sonicated using a 1/8" microprobe tip, the exception being the initial tissue sample which was processed whole. Sonication was performed three times for 5 sec. with 1 min. intervals on ice. The optimal power setting was determined for each sample type (data not shown) and optimal results are presented.|
The yield of lactate dehydrogenase from mouse muscle was very dependent upon the type of homogenization method used. Generally, LDH was more effectively liberated when the tissue was processed with two sequential steps (see figure). One step processing of tissue was effective with the mixer mill, but much less so with glass homogenizers (see below). The rotor-stator was also not overly effective. Two step homogenization methods were more efficient in releasing LDH. Cryogrinding, when combined with other disruption methods, provide very efficient at LDH liberation.
Generally the efficiency of sample disruption as measured by LDH release was greatest in processes that reduced particle size in steps. Cryogenic grinding by itself yielded 30% LDH activity, but when combined with sonication, produced 100% activity. Interestingly, sonication of muscle was an extremely poor method for releasing activity LDH. When cryogrinding preceded glass homogenizer disruption, yields also increased over one step methods using the same homogenizer. It is logical to assume that the cryogrinder reduced the muscle mass to fragments which were further reduced in size by subsequent homogenization steps.
Though a one-step method, bead beating was effective in disrupting tissue for LDH release. Deep well plates with 5/32" grinding balls gave over 50% relative activity while the 15 ml Vial Sets reached over 80%. Though these two methods did not attain the yields of cryogrinding coupled with sonication, they do provide a rapid option for processing many samples. All the two step methods, though efficient, are laborious and low throughput. Bead beating offers a compromise of decent activity with higher throughput.
Photographs of Tissue Lysate (400X)
Tissue lysate from a 15 ml Vial Set processed in a mixer mill was finely pulverized.
Lysate produced from a Dounce homogenizers. Note that significant solid tissue remained in the tube.
Potter Elvehjem homogenizer fragmented tissue, but solid tissue remained in the tube.
Mechanical rotor-stator homogenizer effectively disrupted the whole sample to microscopic fragments.
Cryogenic grinding followed by sonication yields extremely fine homogenate.