Humans are a heterogeneous population and studies comparing populations of humans require a high number of samples for statistical validity. In addition, human samples such as PBMC are precious in that they represent the immune state of an individual at a point in time. Thus, when studies are done to analyze a particular state of the immune response in individuals, such as pre- versus post-vaccination, or along the course of a disease state, once used, the samples can never be replaced. To make the most of human PBMC samples, in particular when patient samples are being used, it is important to not only carefully optimize assays, but additionally be able to maximize the questions that can be addressed with these samples.
Having recently completed a large study involving human patient PBMCs, I encourage the use of high throughput assays systems that allow for a streamlined experimental approach. All of these assays involve 96-well plate based methods and commercially available kits.
Basic Equipment for 96-well Plate Assays:
Multichannel Pipettes are necessary for quickly performing all 96-well plate assays. These come in p1000, p200, p20, and p2 volumes.
96-well Plates: Different types of 96-well plates are available for different assay types. There are various surface coatings including tissue-culture treated polystyrene for cell cultures, uncoated, and others. Plates can have various plate bottom geometries and optical characteristics. For instance there are black plates available for light-sensitive assays. For protocols involving volumes larger then 250ul, there are deep-well plates that carry a 2ml volume per well.
Multichannel Vacuums: Companies such as V&P Scientific offer a multitude of multichannel vacuum manifolds that fit plates of different depths for removing supernatant from wells via vacuum apparatus. Often these will be the proper length such that they don’t touch the well bottom and work well with removing buffers from centrifuged PBMC cell cultures, such as during washing steps for flow-cytometry.
http://www.vp-scientific.com/wands_&_manifolds.php
PBMC subset Purification: For magnetic bead based purification of PBMC populations of interest, Stem Cell Technologies offers a 96-well plate EasyPlate™ EasySep™ Magnet that allows separation of up to 1 x 107 cells per well. Currently only negative or untouched cell isolation methods are supported by this magnetic system due to the larger size of the magnetic beads used in Stem Cell Technologies’ negative isolation kits compared with positive isolation kits.
http://www.stemcell.com/en/Products/All-Products/EasyPlate-EasySep-Magnet.aspx
RNA Isolation: Qiagen offers two kits for 96-well purification of total RNA from cells. The RNeasy 96 Kit and RNeasy Plus 96 Kit. These are 96-well column based platforms which require either a Qiagen vacuum manifold or specialized centrifuge for the protocol. The RNeasy 96 Kit and RNeasy Plus 96 Kit are similar with the RNeasy Plus 96 Kit utilizing an extra set of steps and columns for elimination of genomic DNA. The standard RNeasy 96 Kit protocol does however have an optional step for on-column DNAse digestion, however DNAse is not included in the kit.
RNeasy 96 Kit: http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasy96.aspx
RNeasy Plus 96 Kit:http://www.qiagen.com/products/rneasyplus96kit.aspx
RNA Quantification is much easier if done by 96-well methods than one sample at a time. Life Technologies’ Quant-iT™ RiboGreen® RNA Assay Kit is extremely sensitive but requires a fluorescence-plate reader. Thermo Scientific now has a NanoDrop 8000 UV-Vis Spectrophotometer that quantifies nucleic acid concentrations from 96-well plates.
http://products.invitrogen.com/ivgn/product/R11490
http://www.nanodrop.com/productnd8000overview.aspx
In summary, systematic high-throughput protocols can be developed using 96-well systems such as these and many others. Thus, numerous PBMC samples can be put through multiple experimental procedures in a streamlined manner, maximizing efficiency and minimizing experimental variation. In this way, multiple questions can easily be simultaneously addressed in precious PBMC samples.
