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FAQs

Components

How big are microbubbles?

In particular, we currently use polydisperse microbubbles that are 16–18 um in diameter. Read this blog to learn more.

What are microbubbles made of?

Akadeum’s microbubbles are made of thin shells of glass with a gaseous core. Read this blog to learn more.

How many cells can a microbubble lift?

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.

What is the Separation Buffer?

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.

Does Akadeum have a recommendation for biotin free BSA?

Millipore Sigma Bovine Serum Albumin – Product #A7030

Can Akadeum microbubbles be analyzed by flow cytometry?

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.

Technical – Sample-Related FAQs

My spleens are very large, is it possible to process samples containing >100 million cells in a single separation?

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.

What if my samples are small (<10 million cells)?

When processing samples that are less than 10 million white cells/splenocytes, Akadeum recommends the following protocol changes:

  • Replacing the provided 5 mL centrifuge tubes with low retention 1.5 mL microcentrifuge tubes.
  • Use the same amount of antibody as called for for 10 million cells.
  • Scale down the microbubble number accordingly (but do not use less than 10 μL of microbubbles.
  • When mixing use wide bore 200 μL pipet tips (VWR part no. 37001-186). This minimizes the shearing forces that are produced during mixing, as there is a wider orifice at the end of the pipet tip.

Are Akadeum microbubble enriched samples compatible with flow cytometry?

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.

Can I culture the isolated cells after isolation with Akadeum microbubbles?

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.

I am using an Akadeum mouse kit, do I have to lyse the red cells?

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.

Can I do back to back isolations to improve purity?

Yes, but any improvement in purity will be accompanied by additional loss of desired cells.

Technical – Procedure-Related FAQs

Why is mixing volume 50% of the total volume?

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.

I’m having trouble aspirating the microbubbles, what can I do?

Please watch the video demonstrating aspiration of microbubbles at www.Akadeum.com. Working with microbubbles can take a bit of practice.

What if there are a lot of microbubbles remaining (stuck to the sides of the tube) after aspiration? Is this a problem?

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.

Can I decrease the biotin antibody incubation time?

No. Lower incubation times at 4°C will result in lower purity.

Can I perform the biotin antibody incubation at room temperature for a shorter time?

Akadeum has not determined if shorter room temperature incubations will change product performance so it is not recommended at this time.

After sitting in the refrigerator, all the microbubbles have separated out of solution. Is there something wrong?

This is normal. The solution will need to be mixed well before every use.

How do I know if the microbubbles are mixed enough before using them?

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.

How fast is too fast for mixing the microbubbles?

Trituration should be performed at approximately 0.5 to 1 stroke per second. See the video at www.Akadeum.com for a demonstration.

I’m worried about trituration impacting my cells, is there an alternative mixing method?

Akadeum has found slow mixing by trituration to be the most effective and quickest method for mixing microbubbles with cells.

I don’t have low retention pipet tips; can I use standard tips?

Low retention tips are not required but more microbubbles will adhere to standard tips. This may impact isolation performance.

I don’t have access to a swinging bucket rotor centrifuge, can I still use Akadeum microbubbles?

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.

I don’t have an aspirator/vacuum available in my lab, can I still use Akadeum microbubbles?

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.

Shipping and Storage

How are Akadeum kits shipped?

Kits are shipped on ice packs with overnight delivery.

How should I store components?

Separation Buffer, microbubbles and antibody cocktail should be stored at 4 degrees Celsius.