In particular, we currently use polydisperse microbubbles that are 16–18 um in diameter. Read this blog to learn more.
Akadeum’s microbubbles are made of thin shells of glass with a gaseous core. Read this blog to learn more.
When isolating fully dispersed cells (such as in a Ficoll or leukopheresis PBMC prep), we have seen microbubbles carrying as many as 4 cells. Cells may also serve as a bridge between two microbubbles, significantly increasing the lifting capacity of the overall structure (see fluorescence microscopy example at left, where the microbubbles are blue). Read this blog to learn more.
Ca2+, Mg2+ free Dulbecco’s PBS containing 2mM ETDA, 0.09% sodium azide, and 0.5% bovine serum albumin (BSA). BSA must be biotin free as to not interfere with kit performance.
Millipore Sigma Bovine Serum Albumin – Product #A7030
Yes. Analyzing samples of microbubbles or microbubbles bound to cells can be performed but high concentrations of microbubbles have been found to clog the SIP of various cytometers. Keeping microbubbles in a homogeneous mixture is also difficult as they will quickly rise to the top of the FACS tube during analysis. Futhermore, as the microbubbles are a polydisperse size distribution, it can be difficult to separate microbubbles from microbubbles bound to cells.
No. Samples in excess of 100 million cells should be divided and multiple separations performed. The cells can be recombined afterward. This is a result of the size of the included 5 mL tubes. Samples in excess of 100 million cells require larger volumes of buffer in the wash steps that will exceed the total tube volume. Larger volumes will also change the required mixing volume to be above 1 mL making it more difficult to obtain proper mixing with a standard 1 mL pipet.
When processing samples that are less than 10 million white cells/splenocytes, Akadeum recommends the following protocol changes:
Yes. Residual microbubbles in samples will not interfere with flow cytometry. Microbubbles are larger than many cell types and have very bright side scatter so they will not overlap typical cell populations in a scatter plot. However, the microbubbles fluoresce under certain conditions. This is most prevalent with the use of a UV or violet (405 nm) laser and is dependent on the emission filters used. This does not preclude the use of the Brilliant VioletTM family of dyes, however, it may complicate analysis. Akadeum recommends staining for highly expressed targets in channels excited by UV or violet lasers.
Yes. After removal of the microbubble layer, the remaining cell pellet can be resuspended in any desired buffer and cultured or analyzed with other methods.
Lysing red cells is recommended to increase purity. The antibody cocktails contain a TER119 antibody to capture and remove red cells. However, depending on the sample, it may not be sufficient to remove all the red cells. This may result in lower purity.
Yes, but any improvement in purity will be accompanied by additional loss of desired cells.
This ensures adequate opportunity for the microbubbles to find lower abundance cell types that are targeted. Using a higher percentage (ex. 70%) will perform similarly, however lower mixing volume percentages result in less efficient mixing and poor results.
Please watch the video demonstrating aspiration of microbubbles at www.Akadeum.com. Working with microbubbles can take a bit of practice.
As these microbubbles should only contain cells that are targeted for removal, they should not cause any issues. However, if you wish to remove them, so as to have a cleaner tube, add 1 mL of separation buffer, invert the tube to wash the microbubbles down and centrifuge a second time. Alternatively, the cell pellet can be transferred to a new tube.
No. Lower incubation times at 4°C will result in lower purity.
Akadeum has not determined if shorter room temperature incubations will change product performance so it is not recommended at this time.
This is normal. The solution will need to be mixed well before every use.
The microbubbles should be a homogenous white solution. Rocking the vial, a brief vortex, or mixing with a pipet can all help. After mixing, check if there are any solid patches of white on the side of the vial (there will always be a film of bubbles on the side of the vial). If so, keep mixing. Please watch the video at www.Akadeum.com for additional help.
Trituration should be performed at approximately 0.5 to 1 stroke per second. See the video at www.Akadeum.com for a demonstration.
Akadeum has found slow mixing by trituration to be the most effective and quickest method for mixing microbubbles with cells.
Low retention tips are not required but more microbubbles will adhere to standard tips. This may impact isolation performance.
Microbubbles will still rise to the top in a fixed rotor centrifuge however the microbubble layer will not be even. This can make aspirating more difficult.
Aspiration is the best/easiest way to remove the microbubble layer after centrifugation. Pipetting off the bubble layer is not very effective and pipetting the cell pellet from under the bubble layer is not recommended. Vacuum pumps are available from a variety of vendors.
Kits are shipped on ice packs with overnight delivery.
Separation Buffer, microbubbles and antibody cocktail should be stored at 4 degrees Celsius.
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