FAQ - Microbiology/Bacteriology
1. What are RPM, RCF, and g force and how do I convert between them?
The magnitude of the radial force generated in a centrifuge is expressed relative to the earths gravitational force (g force) and known as the RCF (relative centrifugal field). RCF values are denoted by a numerical number x g (ex. 1,000 x g). It is dependent on the speed of the rotor in revolutions per minute (RPM) and the radius of rotation. Most centrifuges are set to display RPM but have the option to change the readout to RCF. To convert between the two by hand, use the following equation: RCF = 11.18 (r) (rpm/1000)2 Where r = the radius of the rotor in centimeters.
2. What happens if some of the OncoQuick separation medium (blue) is in the upper compartment of the tube?
Spin the tube briefly (15- 30 seconds) at 600 x g. Make sure all of the separation medium is in the lower compartment before adding your sample.
3. What are the advantages of OncoQuick over Ficoll-Paque® or magnetic bead (or other antibody based) separation techniques?
OncoQuick enriches for tumor cells while simultaneously depleting unwanted MonoNuclear Cells (MNCs). Ficoll-Paque (or similar density gradient media) enriches for tumor cells, but does not deplete contaminating MNCs. Therefore, OncoQuick increases the sensitivity and specificity for tumor cells in subsequent applications. Magnetic bead (or other antibody based) separation is as efficient at tumor cell enrichment as OncoQuick, but it is more time consuming and expensive. In addition, positive selection by antibodies can alter the cells internal chemistry and surface receptors.
4. Can I use other specimens besides whole blood with OncoQuick?
Yes. You can use the buffy coat (also called a LRP) (at a 1:1 dilution with a physiological buffer, i.e. PBS) and bone marrow specimens. The depletion of contaminating bone marrow cells is less efficient than removal of blood cells. OncoQuick still has a 10-fold higher depletion of bone marrow cells than Ficoll-Paque.
5. What type of blood collection tube should be used to collect blood for centrifugal separation?
Any tube with an anticoagulant agent (i.e. heparin, citrate, or EDTA). Although EDTA and citrate are preferred, because heparin may stick to the red blood cells and prevent them from losing water.
6. When is it too late to use a blood sample for centrifugal separation experiments (i.e. density gradient, OncoQuick)?
Normally, we suggest you use the sample within 2 hours of the draw. If you store the blood at 2 8º C, it will still yield favorable results if used within 24-48 hours. However, the anticoagulant will cause the cells to swell during extended storage times resulting in increased erythrocyte and leucocyte contamination in the tumor cell fraction.
7. What is the purpose of the plastic membrane in the LeucoSep and Lymphoprep® tubes?
These plastic porous membranes are called frits and help in two ways for rate zonal and barrier techniques. The first way they help is making the addition of your sample to the density gradient medium much faster; you dont have to slowly add the sample to avoid mixing it with the density gradient medium. The second way is generally improved yields and purities, as the discontinuous gradient between your sample and the medium is stringently maintained up until the centrifuge has started.
8. What is under layering and what are its benefits?
Instead of loading a tube with the densest gradient medium first, followed by layers of lighter media, the lightest medium is loaded first. Then the next densest medium is slowly added to the bottom of the tube, floating the lighter medium. The advantage, if done properly, is much sharper bands of the discontinuous gradient. The benefit will be higher yields and greater purities in a rate zonal and barrier techniques.