Protocol for L-selectin-PNAd Interactions under Flow Conditions. This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer.
Protocol for Matrigel Invasion Assays Matirgel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression.
Protocol for Measurement of Cell Adhesion Under Static Conditions This protocol introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity thereby eliminating uncontrolled shear forces and passage of adherent cells through a liquid/air interface. The cells are loaded with a fluorescent dye (6-carboxyfluorescein diacetate) for detection although other methods such as radioactive labels malabels may be used. This protocol is also useful for assaying molecules that promote or inhibit cell adhesion.
Protocol for Microtubule Assembly Purified Tubulin is polymerized and then stabilized with Taxol. The resulting Microtubules are then ready for use in motility assays. Protocol use solutions such as 50 mM Mg-GTP, 50 μl 100 mM GTP, 40 μM Taxol, Taxol Stock,and PM Buffer.
Protocol for Motility using VE-DIC Microscopy Protocol for motility using VE-DIC microscopy. Materials required: Anti-GST (glutathione S-transferase) antibody, PB buffer =10 mM NaPO4 pH 7.2, EGTA, MgCl2, Clarified motor lysate, MTs or Axoneme-MTs,Mg·ATP, VALAP (1 Vasoline: 1 Lanolin: 1 Paraffin, heated gently until melted) and Maxell XR-S120 Black Magnetite Super-VHS tapes or comparable.
Protocol for Needle Assay for Chemotaxis Protocol for needle assay for chemotaxis. Materials required: Zeiss inverted microscope, eppendolf microinjector, eppendolf micromanipulator, Eppendolf femtotips, 100mM cAMP in PM,DB buffer, PM Buffer.
Protocol for Standard Fluorescence Motility Assay Protocol for Standard Fluorescence Motility Assay. Materials requires: Microtubules, Flow Cell, Standard Solutions: BRB80, BRB80T, BRB80CS0.5, BRB80CA, BRB80CT
Purification of viable bovine spermatozoa of normal morphology. Protocol for the recovery of a highly viable fraction for use in fertilization. Protocol uses an iodinated density gradient medium to adjust the density of the raw ejaculate to approx 1.170 g/ml by mixing with a high density medium (Ï >1.26 g/ml) and loading it beneath a discontinuous gradient.
S20 Purification of detergent-insoluble lipid rafts from cells and tissues. Protocol for the isolation of the lipid-rich microdomains of the plasma membrane, notably caveolae and lipid rafts. Methods for the isolation of lipid rafts are based on the insolubility of these structures in the nonionic detergent TritonX-100. Either the intact cells are treated with a detergent-containing solution or a post-nuclear supernatant is prepared from a cell homogenate and then Triton X-100 is added to this supernatant.
Separation of Platelets from Whole Blood This protocol describes how to isolate human platelets from whole blood. Isolated platelets are used for static adhesion assays, for flow chamber assays, flow cytometry measurements, etc.
Telomere Length Determination Protocol Telomere length can vary from strain to strain and is sensitive to a variety of mutants affecting DNA and chromosome function. Includes: Gel Protocol; Southern blot using oligo probe.
Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.
