Analysis Sample Preparation

Sample Properties

Samples must be single-cell suspensions. Cells must be filtered at the cytometer through an 80 µm (or finer) mesh, even if the samples were filtered earlier in the day at the lab bench.  Suitable mesh squares and transfer pipets for filtering are provided by each machine.

If you wish to purchase the same mesh for use in your lab, it's sold by the yard from Elko Filtering Co. (03-80/37) https://www.elkofiltering.com/store/p/434-Nylon-Mesh-80-Micron-Open-Area-37-Width-40-in.aspx.

 

If your samples are prone to clumping and/or clogging the machine, please consider:

  • Diluting your sample
  • Target cell concentration for analyzers is around 106-107 cells/mL (which is 105-106cells/300µL).
  • Cells that rarely clump (filtered lymphocytes) can be SLIGHTLY more concentrated depending on the appropriate event rate of the cytometer, but if you’re having trouble with clogs (or high abort rates), try diluting your sample.  Squeeze bottles of sheath/PBS are provided at each of the cytometers for this purpose.
  • Adding DNAase
  • If there are significant numbers of dead cells present, the DNA released can make cells and cell fragments stick together.
  • Alternatively, figure out why so many cells are dying before fixation and solve that issue. 
  • Adding EDTA
  • If EDTA is incompatible with your protocol, try to use Ca2+ and Mg2+ free buffers where possible.
  • Not over pelleting your cells
  • Use the minimum force and time required to collect your cells EACH TIME.
  • Getting rid of red blood cells and debris
  • Options range from ACK lysis buffer to density gradients such as Ficoll.

 

Minimum Sample Volumes

Analyzer

Sample Tube Formats

Minimum Sample Volume

Aurora

PS or PP 12x75mm tubes

“bullet” tube inside 12x75mm tube

96 well plate

0.1 mL

Attune

PS or PP 12x75mm tubes

“bullet” tube inside 12x75mm tube

96 well (only)

90 µL (tube), 50 µL (plate)

Fortessa

PS 12x75 mm tubes ONLY

“bullet” tube inside 12x75mm tube

0.3 mL

LSR II

PS 12x75 mm tubes ONLY

“bullet” tube inside 12x75mm tube

0.3 mL

ImageStream

1.5 – 2 ml microcentrifuge tube

96 well (only)

20 µL (tube), 50 µL (plate)

Abbreviations:  PP = polypropylene, PS = polystyrene

 

Fortessa and LSR II are especially picky about their sample tubes.  They must be intact (no cracks) and made from polystyrene only.  Falcon catalog numbers: 352052, 352054, or 352058 work quite well.  Transfer of samples may be required if using inappropriate tubes.

 

Safety Concerns

Samples containing potential bio-hazards are recommended to be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition. If the user has to run those samples without fixation, he/she must follow the instructions in the section: “Unfixed BSL2/2+ Specimens on STI Fortessa in BST Room S756.”  Otherwise, samples containing known biohazard or infectious agents which are above BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition.

 

Users cannot discard tubes with cells in fixative solution (e.g. paraformaldehyde) in the Core. All samples containing either fixative or unfixed human, non-human primate, or virally transduced/infected cells must be disposed in your own laboratory by following EH&S recommendations.

 

If you have any questions about your particular experiment, please ask a member of the Unified Flow Core for guidance.  

 

Unfixed BSL2/2+ Specimens on STI Fortessa in BST Room S756

Aerosols can be generated when taking the sample tube off from the sample injection port (SIP) on the LSR II or Fortessa.  Since some users may need to run potentially bio-hazardous specimens without fixation, they are asked to use the Fortessa inside the class II biosafety cabinet in room S376, which is a Biosafety Level 2 (BSL2) certified room. By following the BSL2 requirements from EH&S, we request that samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition.  If a user has to run a potentially bio-hazardous specimen without fixation, such as unfixed human, non-human primate cells, or virally-transduced or infected cells, in addition to following our general guidelines for using LSR II and Fortessa, he/she must also do the following:

1.       Before starting the experiment, users need to TURN ON the hood.

2.       After finishing the experiment, the user MUST decontaminate the working area by spraying Viraguard or Cidehol 70 around and under the SIP areas and let it stay for at least 10 min. 

3.       Then follow the “cleaning and shut down procedure” by running 10% Bleach tube for 10 min instead of 5 min.  Water is then run for 5 min to rinse away the bleach.

4.       Before the user leaves the lab, he/she must wipe the areas that have been sprayed with Viraguard or Cidehol 70, using a paper towel

5.       TURN OFF the hood.

If you have any questions about your particular experiment, please ask a member of the Unified Flow Core for guidance.