Scale the combination for the number of samples being stained. (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific info than assays such as a total blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining process is simple, requires only 100 l of new whole blood, and takes approximately 45 moments, making it feasible for standard blood-processing labs to perform. It is adapted from your BD Trucount VTX-2337 tube technical data sheet (version 8/2010). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to Rabbit Polyclonal to SLC10A7 the site processing labs. Stained tubes can be fixed and freezing for shipment to the central assay laboratory for multicolor circulation cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to VTX-2337 assess activation claims of specific cell types of interest. In this statement, we demonstrate VTX-2337 the procedure used by blood-processing lab technicians to perform staining on new whole blood and the steps to analyze these stained samples at a central assay laboratory assisting a multicenter medical trial. The video details the procedure since it is performed in the context of a medical trial blood draw in the HIV Vaccine Tests Network (HVTN). To protect the fluorophore-conjugated antibodies from light, perform all methods in a bio-safety cabinet with the light off. 1. Antibody Staining Panel Preparation The antibody staining panel can be found in Table 1. Antibody concentration should be defined by titration with whole blood and using the same circulation cytometry products and procedures that’ll be used to acquire the stained phenotyping samples. Once appropriate staining titers are identified, combine all antibodies into a solitary mixture inside a lock-cap tube. Add flow wash buffer (Dulbecco’s PBS with 2% warmth inactivated fetal bovine serum) to bring the total volume to 100 l. Level the combination for the number of samples becoming stained. This combination can be stored at 4 C for up to eight weeks. 2. Staining If the blood collected is to be used for additional purposes VTX-2337 in addition to this assay, arranged an aliquot aside while more time-sensitive methods are performed on the remaining blood. The aliquot can be stored at space temp for up to 4 hr after venipuncture without significant cell loss. Verify that there is an intact bead pellet at the bottom of the Trucount tube and label the tube to identify the sample becoming stained. In HVTN medical trials, the Laboratory Data Management System (Frontier Technology and Technology Study Basis; Amherst, NY) is used to label and track stained samples. Record the lot figures and expiration times of all reagents. Record the Trucount tube bead count quantity provided by the manufacturer within the bag of tubes; make sure that the lot quantity within the bag matches the lot quantity within the tube. Use reverse pipetting to accurately pipette 100 l of whole blood into the Trucount tube, just above the metallic retainer. Avoid smearing blood down the side of the tube. Using regular (ahead) pipetting technique, pipette 100 l of the combined antibody staining panel (See Table 1) into the Trucount.