Frequently Asked Questions
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When using Product 11191, Histopaque®-1119, the mononuclear cell layers forms as expected, but the layer with the polymorphonuclear (eg, neutrophils) does not form properly. Why?
Technique is much more important with Histopaque® 1119. The published product insert calls for layering in the 1119 first, then adding a layer of 1077 and then layering the blood. If there is any mixing between the 1077 and the 1119, the procedure does not work properly. Customers can check for the integrity of their interface by checking for Schlieren optics. Any swirling or mixing should be evident when holding the tube up against a light source, such as a ceiling light or towards a window. If the interface was properly prepared, there should be a sharply demarcated line. If swirling is present, the user should start over. It is also important to use the gradient as soon as it is formed. There are no chemical differences between 1077 and 1119. They are the same product, only different densities. If left together for any length of time the two products will start diffusing together. When this happens the percent recovery becomes unacceptable. The gradients must be used immediately after preparation - without delay. Creating the gradient requires the steadiest of hands. Until you master the technique, limit the number of samples you attempt to process in one run. After gaining experience, the number of samples processed at one time can be increased.
Some customers prefer to underlay. This protocol starts with 1077 in the test tube. Then a long metal cannula or a 9.5" Pasteur pipet is used to introduce the 1119 into the very bottom of the test tube. Care must be taken not to introduce any air bubbles. The air bubbles will destroy the interface. Load ~2.9 mls of 1119 into a 9.5" Pasteur pipette. With an electric pipet aid, add very slowly to the bottom of the tube. With this technique the Histopaque® 1077 is slowly pushed upwards and displaced by the Histopaque® 1119 When done properly very sharp gradients are possible. Electric pipet aids are useful when overlaying and underlaying Histopaque®. The release is dependent upon how slow or fast the dispensing button is applied.
Regardless of the technique employed, it is imperative to introduce the Histopaque® slowly and smoothly. Users trying to do this with a 10 ml pipet hooked up to a blue rubber bulb are often going to experience problems. When dispensing, tilt the tube slightly, touch the pipet to the Histopaque® already placed in the centrifuge tube, draw the meniscus up the side of the tube 4 or 5 mm and then allow the Histopaque® to be released. No mixing should occur. If dispensed too quickly, the Histopaque®/blood will mix with the lower layer.
When releasing the Histopaque® into the centrifuge tube it is beneficial to slant the tube at a 45 degree angle. The Histopaque® is then slowly released. When complete there should be an absolutely sharp, visible line at the interface between the Histopaque® 1077 and the Histopaque® 1119.
It is imperative the blood and Histopaque® all be at room temperature. Having either the blood or Histopaque® cold could well cause the red cell contamination to increase and the cell recovery to become unacceptable.
Another tip involves the measurement of the radius for your centrifuge. The measurement should be from the center of the spindle to the bottom of the centrifuge tube (when in the swung out position). Calculating the radius to the top or the middle of the centrifuge tube could also account for separation problems. It is debatable which method is correct - top, bottom or middle. Sigma uses the bottom of the centrifuge measurement. Some centrifuges automatically calculate rpm. Check the owner's manual for your centrifuge to determine how the rpm calculation is performed. Not all manufacuters use the method recommended by Sigma-Aldrich.
If you are collecting your blood with a syringe, switch over to a vacuum tube. Use the freshest blood possible, but do allow the blood to reach room temperature.
Can the procedure for using Product 11191, Histopaque®-1119, be used successfully with species such as rats and mice?
The Histopaque 1119-1 procedure was developed for use with human blood. Due to the lower number of circulating neutrophils in rat or mouse peripheral blood, the technique tends not to work as well with rats or mice.
When using Product 11191, Histopaque®-1119, what is the expected percent recovery for the band containing neutrophils.
No percent recovery data has been generated. Due to the importance of technique with the Histopaque 1119-1, percent recovery varies with the skill of the operator.
The Product 11191, Histopaque®-1119, froze in transit. Is the product still suitable for use?
Mix the bottles upon receipt several times to ensure the product is uniform. The products are shipped ambient and gradients in situ can form if the solution freezes during transport. As long as the bottle has not broken and no precipitate is noted, the product is suitable for use after slowly thawing and mixing.
Can the procedure for Product 11191, Histopaque®-1119, be performed using the various Accuspin products?
No. Accuspin products are intended for use with just a single density gradient - such as Histopaque® 1077.