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

• 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.

• 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.  

• Options range from ACK lysis buffer to density gradients such as Ficoll.

Minimum Sample Volumes

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

Sample Properties

Cells (if <40 μm) in all samples and controls must be filtered through a <40 μm nylon mesh filter to prevent clogging of the instrument right before they are brought into the sorting room or right before they are introduced into the cell sorter even if the cells have been filtered earlier before staining in the user’s lab.

Samples can be in any of the following tubes: 12 x 75mm, 15 mL, or 1.5 mL .

The concentration of lymphocyte-like cells can be up to 5 x 10⁷ cells/mL.  

For a multicolor experiment, the user must bring an unstained control tube and a single-color stained samples (ideally >5% positive) to use for compensation.  The user should consult Core staff for the controls required for the instrument set up if there is any confusion.

Users need to bring collection container(s) which can be any of the following options:

• Tubes: 12 x 75mm, 15 mL, or 1.5-2 mL.  For 4-way sorting, only 12 x 75mm or 1.5-2mL tubes can be used.
• Plates: 6-,12-,24-,48-, 96- (including PCR plate), 384-well, or custom size
• Trays/dishes or slides

The collection container should be pre-filled with the desired volume of collection medium. 

If this is a new sort:

• Please bring extra sample buffer in case sample dilution is required.
• Please bring extra collection tubes/media, especially if a large number of cells is being collected and/or the sort requires a larger nozzle size.  (The droplet of sheath fluid (PBS) around each cell will add volume to your collected sample.)

Please comment on any other special handling requirements for your samples, i.e. light sensitivity, tendency to clump, keep on ice, etc. The more we know about your experiment, the better the results.

Collection Volumes to Expect

Safety Concerns

No radioactive materials are allowed in the facility.

Unfixed samples known to contain BSL-3 pathogens or unfixed samples recommended to sort at BSL3 will not be accepted. The Operational Director will make the decision concerning sorting of other known infectious agents on an individual basis in consultation with the EH&S Biosafety Officer when necessary.

Criteria for samples containing potential bio-hazards, such as unfixed human, non-human primate, virally-transduced or infected cells:

• Sample must be contained in a leak-proof container with a secure lid and be clearly labeled with a sample identifier and a biohazard symbol, if needed.
• Sample must be clump free and in an appropriate tube for sorting (12 x 75 snap cap tube or 15 mL tube with cap).
• Sample must not contaminate the outside of the tube.

Please indicate biosafety concerns when scheduling the sort. 

Viability Concerns

Sorting can be very stressful to cells.  By far the biggest stressors are the pressure changes experienced by the cell as its droplet goes through the nozzle.  Larger diameter nozzles sort more gently but samples must be run more slowly due to the lower pressure.  Thus there is a practical tradeoff between the time spent in the sorter (and outside of growth media/warmth) and the pressures required for an efficient sort.

Other things that can be done to preserve cell viability/sort more efficiently

Liquid in the collection tube

• Cells that hit a dry surface will almost always die.  Eventually enough dead cells and sheath buffer (PBS) will build up to give the later cells a chance to survive, but why lose cells unnecessarily???

• Many protocols recommend a lower serum concentration during sorting, and compensate with a higher serum concentration in the collection media.

• Most cell media buffers are bicarbonate based, and will tend to be alkaline outside the incubator’s 5% CO₂ atmosphere.  If your sorts and benchwork take a long time outside the hood, consider adding sterile HEPES buffer to your media.  Especially if your red media looks a little more pink/purple.

• Some cells (not all) will benefit from being kept cool until they can be returned to growth media and the incubator.
  • Our sorters have optional cooling jackets for sample collection.  Simply request one in the comment section when you schedule the sort.
  • If you have several samples or they run slowly, leave an ice bucket with us to keep your waiting samples cool.

• Consider using a method such as magnetic beads to pull out cell populations you are NOT interested in, so that your cells of interest comprise a larger percentage of the remaining sample.  The sort will run faster, reducing cell stress and sort costs.

• This makes sure the cells only live cells are sorted.
• We have observed that larger cells do take up some live/dead dye, although not as much as dead cells.  Sometimes with larger cells you’ll want to collect the dim cells along with the negative cells, and exclude only the bright dead ones.
  • You can always do a control wherein you take a population of living cells, heat kill ½ of them, recombine and stain with your favorite viability dye.  This should give you a clear indication where to gate.

• Some commercial flow cytometry buffers contain sodium azide.  Make sure there is no azide in any buffers where the cells are expected to live.

Sample Properties

Cells are recommended to be suspended into 1 x PBS with no more than 2% FBS at the concentration of 1 million/50µL.  Minimum volume of each sample is 20µL (50µL if using a 96 well plate).

Samples must be a single-cell suspension. If the user experiences the significant sample clumping, cells need to be filtered through 80µm (or less) Nylon mesh (Small Parts, Inc. cat# CMN-0074).  Suitable mesh is provided at the ImageStream desk.

For manual acquisition, the sample must be in a 1.5-2 mL microcentrifuge tube (siliconized, e.g. Sigma, cat# T4816).  For the auto-sampler, the samples must be in a 96-well plate (e.g. Corning, cat#3790).  The wells can have round, flat, or pointed bottoms. A pierceable cover (e.g. X-Pierce, cat# XP-100) is recommended for the plate. 

Safety Concerns

No radioactive materials are allowed in the facility.

Samples containing known biohazard or infectious agents which are BSL2/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, please discuss your specific experimental situation with Unified Flow Core personnel to work out an appropriate safety protocol consistent with EH&S guidelines, IN ADVANCE.

Detailed protocols for sample prep can be found at: https://canopybiosciences.com/zs-support/bench-work-protocols-tissue-samples/

Generally, you need to section tissue at 5-7µm onto a coverslip (provided by Unified Flow Core or through Canopy).  If this is frozen tissue (FF), be sure to FIX the tissue before mounting the coverslip onto the chip.  If this is paraffin embedded tissue (FFPE), be sure to BAKE, DEPARAFFINIZE, and PERFORM ANTIGEN RETRIEVAL steps before mounting the coverslip onto the chip.

Do NOT prestain your tissue before bringing it to be scanned.  The procedure is to photobleach the tissue first, acquire a background image, THEN stain, and acquire.

When performing subsequent staining cycles please keep these guidelines in mind:

• It's a good idea to photobleach and acquire background images within a few days of the NEXT staining cycle. 
• Make sure your tissue is properly photobleached in each channel you intend to stain in before applying the next stain.
• Do not acquire data when the sample is in storage buffer; always switch to wash buffer before scanning or bleaching.

Reference:
https://canopybiosciences.com/zs-support

Flow Cytometry Protocols

General Tips for Sample Preparation