BIO-SPECIMEN PROCESSING

The following tasks are covered under this service category:

Blood Sample Processing

Separation of lymphocytes from whole blood by differential centrifugation thru Ficoll-Hypaque or Lymphocyte Separation Medium (LSM) or in Leukoprep tubes. The RBCs pellet down while lymphocytes are suspended as a "buffy coat" between the LSM and plasma or between the gel of the Leukoprep tube and the plasma. Plasma can be removed from the top and saved for testing. The "buffy coat" can then be removed by gentle suction with a pasteur pipette. Cells are then washed and used for further analysis or for cryopreservation.

B and T Lymphocyte Separation

Enriched B cell populations are obtained by depletion of T-lymphocytes by using F(ab')2 of Pan-T coated flasks and antibody coated magnetic beads.

Monocyte Depletion

Techniques for removal of macrophages and monocytes from PBMCs also involves plating of cells in tissue culture flasks coated with human AB serum overnight. The monocytes attach to the surface and other lymphocytes are removed by pipetting out the rest of the cell suspension.

Specimen aliquoting related tasks

These tasks are usually initiated by the client at the time of receipt of samples or shipping out or testing an aliquot. We use screw cap plastic vials, usually with 0.5 ml to 2 ml capacity. These tubes are sterile and can withstand low temperatures of up to minus100oC for a minimum of 10 years. Caps for these tubes are provided with a rubber leak proof seal ring. We use digital print-labeling device that can be linked to a desktop computer and has ability to generate regular print labels, bar-coded labels or a combination of both. Labels that are cold resistant or capable of sticking to frozen vials are kept in stock for any anticipated tasks. The print portion of the sample labels is secured with a transparent tape part which renders labels impervious to water and stable in -70oC storage and in liquid phase nitrogen storage.

Cryopreservation

For long term storage in liquid nitrogen, peripheral blood mononuclear cells and tissue culture cells are cryopreserved in freezing medium containing 10% DMSO and 10-20% fetal calf serum in growth medium characteristic for that cell type. The protocol has been developed to yield more than 95% viability on revival of such cells.

DNA Isolation

KTI has developed protocols for isolation of genomic DNA from tissue culture cells or tissues including blood samples, and of both plasmid and genomic DNA from bacterial cultures. In general, the protocols involve lysing of cells followed by 3-4 phenol-chloroform extractions, and ethanol-salt precipitation. If desired, the pelleted DNA can then be purified by 2 bands of CSCl gradients. Alternatively, Qiagen resin columns are used to isolate the DNA from the lysed cells. These columns yield purity of DNA equivalent to rounds of purification on CSCl gradients. The end product can be used for DNA minipreps as well as for large amounts of DNA.

RNA Isolation

Procedures have been developed to isolate total RNA or mRNA from cultured mammalian cells as well as bacterial cultures. Like DNA isolation procedures, cell lysis in guanidine isothiocyanate is followed by CSCl gradient centrifugation to get the RNA pellet. The pellet is dissolved in Guanidine hydrochloride, precipitated in acetic acid-ethanol, extracted with water, reprecipitated in potassium acetate- ethanol and resuspended in water. Alternatively, Qiagen resin column can be used for quick efficient isolation of RNA.