Prepare the SOP for density gradient centrifugation of animal cell lysate.
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Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.
Studies of the human immune system often involve isolation of peripheral blood mononuclear cells (PBMCs), and appropriate protocols for isolation and storage of PBMCs are important to facilitate this research. Here, we provide standard operating protocols for the acquisition, isolation, freezing, storage, and thawing of PBMC.
Obtaining live blood cells for analysis requires the use of anticoagulants during blood collection. The choice of collection tube depends on the ultimate downstream applications. Commonly used anticoagulants include ethylenediaminetetraacetic acid (EDTA) (in purple or lavender top vacutainers), acid citrate dextrose (ACD) (yellow top vacutainers), sodium citrate (light blue top vacutainers), and sodium heparin (green top vacutainers). Because additives used in blood collection can affect the suitability of the cells for downstream assays, careful consideration should be made in deciding which type of vacutainers to use for blood collection. For example, EDTA has been shown to interfere with antigen-specific T-cell responses by some,1 but not others2 when compared with blood collected using ACD or heparin. Sodium heparin binds to DNA and interferes with many enzymes used for molecular analyses of DNA therefore should be avoided when the cells are destined for gene expression analyses.3 Cyto-Chex tubes contain fixatives that preserve the blood cells for later flow cytometric analysis; however, fixatives in Cyto-Chex tubes kill cells and can also destroy certain antigens for flow cytometric detection.4
The most commonly used method to isolate PBMCs from blood is density gradient centrifugation using Ficoll. To achieve high yield and purity of cells with reproducibility, multiple parameters need to be considered, such as blood storage condition before processing, choice of blood diluent, Ficoll underlay/overlay technique, and centrifugation conditions. Special blood collection tubes are also available to reduce the variability, such as cell preparation tubes (CPT), prepackaged with a polyester gel and density gradient medium so that they can be directly centrifuged to separate PBMCs from RBC and granulocytes and plasma. The CPT tubes are available with the various anticoagulants as described above.