The Sigma Aldrich Accuspin System-Histopaque 1077 is a proven system for separating lymphocytes and other mononuclear cells from whole blood. The separated mononuclear cells can then be used for downstream applications and analysis such as flow cytometry, HLA typing, and many others.
The effectiveness of the accuspin-system-histopaque 1077 is due to the ACCUSPIN™ Tube which is specially designed with two chambers separated by a porous high density polyethylene barrier (“frit”). Anticoagulated whole blood may be added to the top chamber of the tube without risk of mixing with the HISTOPAQUE®-1077 in the lower chamber under the frit. On centrifugation the whole blood descends through the “frit” to contact with the HISTOPAQUE®-1077. The elements of greater density displace a volume of HISTOPAQUE®-1077 above the “frit” giving a clear separation of the blood components. The erythrocytes aggregate and the granulocytes become slightly hypertonic, increasing their sedimentation rate, resulting in pelleting at the bottom of the ACCUSPIN™ Tube. Lymphocytes and other mononuclear cells remain at the plasma-HISTOPAQUE®-1077 interface. This dense band of mononuclear cells may be collected by pouring off the contents of the upper chamber or by means of a pipet. Erythrocyte contamination is avoided due to the barrier between the chambers.
The Accuspin product line includes 5 different kits. Each kit is unique based on separation capacity, inclusion of Histopaque 1077, and number of polypropylene radiation sterilized tubes fitted with a high density polyethylene barrier included.
Product A1805 includes 20-12 milliliter sample tubes each fitted with a high density polyethylene barrier secured at the 3 milliliter mark.
Product A6929 contains 40-12 milliliter sample tubes, again each tube is fitted with a high density polyethylene barrier secured at the 3 milliliter mark but also include 3 milliliters of Histopaque 1077.
Both products A1805 and A6929 have the capacity to separate between 3 and 6 milliliters of whole blood.
Product A2055 includes 10-50 milliliter sample tubes each fitted with the high density polyethylene barrier secured at the 15 milliliter mark.
Product A7054 contains 12-50 milliliter tubes, each tube is fitted with the high density polyethylene barrier secured at the 15 milliliter mark but also include 15 milliliters of Histopaque 1077.
Product A0561 is the same as A7054 but contains 100-50 milliliter tubes.
Products A2055, A7054, and A0561 can separate between 15 and 30 milliliters of whole blood.
Product number A1805 will be used as an example for the separation of whole blood using the Accuspin system.
It’s important to remember that when working with human source substances that proper precautions be exercised.
To begin, bring the Histopaque 1077 to room temperature.
Once at room temperature pipet 3 milliliters of Histopaque into the upper chamber of each of the 12 milliliter accuspin tubes.
Centrifuge the tubes at 800gs for 30 seconds at room temperature to ensure all the Histopaque 1077 is below the high density polyethylene barrier.
Once the tubes containing the histopaque 1077 have been prepared it would be a good time to label the tubes if your experiment requires it.
Now sample can be added to the accuspin tubes. Freely pour 3.0 to 6.0 ml of fresh anticoagulated whole blood into the upper chamber of each ACCUSPIN tube.
Next centrifuge the accuspin tubes containing the sample at 1000 x g, maintaining room temperature, for 10 minutes or centrifuge at 800 x g, maintaining room temperature, for 15 minutes. Be sure the brake is turned off on your centrifuge for this step.
After centrifugation, Erythrocytes and granulocytes should pellet to the bottom of the ACCUSPIN™ tube. Mononuclear cells should band at the interface between the HISTOPAQUE®-1077 and the plasma.
To recover the mononuclear cells carefully aspirate the plasma layer, with a Pasteur pipet, to within 0.5 cm of the opaque interface containing mononuclear cells.
Then transfer the mononuclear band, with a Pasteur pipet, into a clean centrifuge tube.
Alternatively, the mononuclear band can be poured off into a clean centrifuge tube.
At this point the mononuclear band can be washed to prepare the cells for downstream analysis.
D. Benefits and Troubleshooting
The Accuspin System-Histopaque 1077’s unique design greatly increases the yield of lymphocytes and other mononuclear cells compared to using Histopaque 1077 alone, as this example shows. Within seconds of adding whole blood to the tube containing histopaque alone the whole blood begins precipitating into the histopaque 1077 while in the Accuspin tube the blood and histopaque 1077 remain separated until centrifugation begins.
The technical bulletin for the Accuspin System-Histopaque 1077 contains a number of useful tips for maximizing mononuclear cell recovery from whole blood and bone marrow.