Sorting Guidelines/Sample Prep
Our BDAria IIIs are 5 laser cytometers, hybrid cuvette based sorters (as opposed to traditional jet in air such as the Influx or Astrios). They can sort for yield or purity, and do single cell or bulk sorts (1 – 4 way sort). Please note that for using 15 ml tubes we can only do a 1- 2 way sort. We can perform enrichment and then a purity mode if the sample volume is large. BD has a technical sorting application note that can be read here.
Sample Processing: prepare your sample in as short a time as possible to decrease the stress on the cells.
Transportation of the cells: Cells should be transported on ice, protected from light. We suggest the use of an ice box.
Which fluorochromes can be measured: The flow cytometers can use a variety of fluorochromes and the configuration of the sorters are detailed for each instrument. Spectrum Viewers are available online by different companies.
Agitation and Temperature: The sorter allows for agitation of the cells to prevent clumping and we can control the temperature of the sample tube and collection tube. Please let the Core staff know if this is required or can affect the cells.
Do you need very high purity sorted cells: If you have a large volume and need very high purity or if the starting frequency of the wanted cell is low we can perform an enrichment sort and resort the cells in purity mode.
A suggested starting concentration is:
Small cells such as Lymphocytes, Thymocytes, Natural Killer cells: 8-30 x10E6 per mL
Larger Mammalian cell lines such as CHO, A549: 5-10X10E per mL
Bacteria: diluted to prevent “swarms” per mL
If you are uncertain what concentration of cells to bring, we recommend keeping the cells concentrated and bringing extra buffer so we can dilute them. If the concentration is too high, the co-incident rate increases and the sort efficiency decreases. Furthermore there is a high chance of clogging the machine which means we have to clean the machine and re-do the drop delay which can take up to 30 minutes.
A Short Note On :
Coincidence Aborts: The sorter decides if the cell should be sorted or sent to the waste is based on the events in real time. If the event is too close to a possible undesired event, the machine will abort (not sort) the event to ensure purity. If recovery is a concern, aborts tend to be a problem. There are other sort modes that support recovery rather than purity, so if total cell numbers are more important than purity and should be told to the Core staff if this is desired.
Signal CVs and Sensitivity: If the sample is dilute, to get them to run at a reasonable rate the sample differential must be increased. However if the differential is too high, the CV’s (coefficients of variation) increase leading to a loss of resolution and lower sensitivity. When separating two close populations, the CV becomes more important.
Resolution of populations: Measurements in flow cytometry are based on probabilities that are derived at a number of points. When the dim “positive” population is sorted it maybe at the high end of the negative population distribution. We will try to accommodate this however it is important to note that in sorting sort dim populations from negatives at high purity one must compromise yield and set very conservative sort regions.
Gating Logics: The DIVA software like many of the commercially available data analysis software allows for logical Boolean operations such as “and, or, not” arguments and “Parent to Child” gating. Please contact the core staff prior to the sort to discuss this if you are sorting based on this.
Requesting a Sort: Please contact Core Staff to request a sort.
It is the User’s responsibility to inform the Core of the samples safety levels and precautions needed.
We need at least 48 hrs notification of a sort as we need to prepare the sheath fluid. Please let us know if you need to cancel a sort as soon as possible
Size we can sort- 200 nm to 70 um particles.
We require to know the specific cells/particles you are going to sort so we can choose the correct nozzle and pressure. Please be as specific on the sort forms as possible.
Pre – Sort Heath and Safety Setup Specifics
Radioactively labelled, flammable, abnormal prion infected samples, or BSL3 samples are not allowed at our facility. Refer to the U of A Environment, Health and Safety Biosafety Guidelines for risk and hazard assessments.
The Core Manager has the right to deny sort requests, which have no prior specific Safety approval, if approved experimental protocols are modified in any way, or if conditions have not been clearly disclosed by the user.
1. Pre-screening for Live Primary Human Sorts
Requests for live cell sorting of primary human samples must be accompanied by screening results and be negative for Hepatitis B, C and HIV infections. User/PI must confirm that the patient had no known history of active Mycobacterium tuberculosis infection and no known exposure to the following agents: Neisseria meningitidis, Chlamydia psittacci, Coxiella burnetii, HTLV-1, 2, LCMV, vesicular stomatitis virus. The use of such reagents in live samples is to be disclosed on the “Annual Disclosure form” and is communicated with the Core Staff prior to appointment requests
￼2. Other Experiments
Other experiments may use viral agents (ie. EBV, vaccinia, HTLV, lentivirus, nanoparticle etc.) to manipulate cells, which may require the delegation of designated (vaccinated, non-pregnant, EBV+, etc.) staff. The use of such reagents in live samples is disclosed on the “Annual Disclosure and Sorting Request form” and is communicated with the Core Staff prior to appointment requests. Note: If your I model is considered BSL2 then your sample is also considered BSL2.
Buffers and Practical Considerations
Bringing your samples to the sort
The correct sort buffer is critical for maintaining viable cells during the sorting process. Normal cell culture medium is not ideal as the pH changes to more alkaline when on the bench and the calcium chloride in most culture media are incompatible with the phosphate component of the Sheath fluid buffer. So just like in live microscope imaging you must modify the buffer and the below are suggested starting points.
- Use cell culture, sterile Ca2+, Mg2+ free PBS or HBSS.
- Add the 10-25mM of HEPES, pH range 6.8 – 8.2 (for example: HEPES buffer solution 1M in H2O, Sigma-Aldrich Co., Cat# 83264-100ML-F) to your buffer. This will increase the buffer capacity of the sample buffer which is helpful in high pressure that occurs during the cell sorting procedure.
- Addition of 1-2% FCS or BSA.
- For cell lines and adherent cells the addition of 0.5 – 5 mM EDTA is recommended.
- Addition of DNase I (0.1 mg/mL (or 200 Kunitz units/mL) and incubating for 15 minutes at room temperature prior to proceeding with downstream applications if there is a lot of cell death in your sample.
- Samples must be single cell suspension – Filter your samples through a 30 – 70 um nylon mesh filter depending on the cell type.
- Use anti-clumping agents, such as Accumax/Accutase, soybean trypsin inhibitor in place of serum for inactivation of trypsin.
- We suggest for most cell types to bring them on ice.
Collecting your sorted samples
- You must provide tubes for collecting the sorted cells with media to place into the capture tubes. We suggest to gently pellet and wash all cells after sort and replenish with fresh media prior to placing back into culture or other post sort design.
Suggested media in tubes are:
- 100% serum
- PBS with HEPES and 50% serum
- HBSS with 50% serum
Do not leave the cells in the tubes for a significant amount of time after sorting . While we clean the sample delivery before each sort and sterilize the entire sheath fluid path every week (sheath fluid goes through an in-line 0.2um filter as well), our environment is aseptic and not sterile. It is recommended to culture sorted cells in antibiotic medium e.g penicillin/streptomycin or 50μg/ml gentamicin for a week.
What to Bring
Its is the Users responsibility to bring the cells and controls in the correct medium, collection tubes/plates (FACS Tubes should be no more than 2/3 full) and medium.
1. Cells in standard FACS TUBES
Min. of 1x10e6 cells/ml, and a minimum volume of 500 uL
- Provide gating controls – Fluorescent Minus One (FMO) controls, is the most accurate way to evaluate the background in a channel and set sorting gates.
- Provide an unstained control.
- All separate single color stained cell/beads controls and unstained controls
Live/Dead Amide dyes – refer to the protocol from the vendor. A good way to kill cells is to heat shock them for 10 minutes at 72c
Clontech had compensation beads for GFP and mCherry.
Note : Use of cells and beads in the same compensation matrix. This mean that if you have a functional dye such as TMRM or PI which there are no compensation beads for and you are staining for surface markers, RNA, mRNA, microRNA etc cells can be used in the compensation matrix.
Tubes/Carrier to Sort Into
Tube size depends on the expected number of post-sorted cells and the type of sort.
- Screw Cap Micro Tube (up to 2.0 ml)
- 1.5 ml – sterile (e.g. Cat#3616-875-300, VWR)
- 1.5 ml – low retention tubes (e.g. Cat# 3039-560-000, VWR)
- 2.0 ml – sterile (e.g Cat#3666-875-300, VWR)
- FACS tubes 12 x 75 mm tubes (up to 4.5 ml)
Sort using ACDU (automated cell deposition unit):
- Multiwell plates:
- 96 well plate or strips, flat or round bottom
- 24 well plate
- PCR Tubes in stripes
- Microscope slides
- If we have to customize the set-up please bring the plate a few days before hand for us to do so.
Tube special characteristics depend on the sorting purpose:
- For sterile sort tubes should be also sterile
- For RNA work tubes should be RNAse free
- For Western blot tubes should not be treated with external protein