FACSAria(BD) Tips on Cell Preparation
Multiple factors need to be considered when analysing flow cytometry data, especially when sorting cells, the following tips have been compliled to help resolve common problems with protein concentration, buffering, cell conditions, viability and autofluorescence.
| Protein Concentration (Top) | |
| Light Scattering | Light scatter is related to the
magnitude
of the
refractive mismatch of the particle to the surrounding medium coupled
with the surface size of the refractive mismatch. Any refractive mismatches between sample buffer and surrounding sheath will play a role in scatter measurements. High protein concentrations in the sample buffer can create this Schlieren Effect and result in distortions in the light scatter. |
 | |
| Healthy Cells | Samples should be prepared for
sorting
with the
minimum supplementation of protein required to maintain viability.
Ideally, less than 2% FCS or substitute 0.2-1% BSA instead, as the data
quality should be significantly improved using a lower concentration of
BSA. The media can be any buffered isotonic salt solution (such as PBS) but calcium and magnesium free Hanks Balanced Salt Solution (HBSS) containing 10 mM HEPES is recommended. Addition of 0.5% EDTA may prevent cell aggregation. However, in rare instances sub-optimal levels of protein may be required to insure cell viability. |
| Media
Supplements (Top) |
|
| Phenol Red | Phenol Red is often added to culture medium as pH indicator. The colour changing from orange @ pH7.4 to red under alkali conditions and yellow under acidification. As well as effecting the refractive index of the sample medium, the dye has fluorescent properties that may interfer with the detection of standard fluorophores used for cell identification. Therefore, wash cells and avoid phenol red to minimise background fluorescence. |
| |
| Sample Media | For optimal resolution it would be best to use the same buffer for your sample and the sheath. However, many cell types are sensitive to the effects of pH and viability may suffer if sample medium is not adequately bufferd. In culture medium this is usually achieved by bicarbonate/CO2 buffering. However, during sorting cells are exposed to increased pressure and the partial pressure of CO2 increases and the pH of the sample medium is reduced. To counter this effect use a phenol red-free medium containing the organic zwitterionic buffer HEPES or supplement PBS or HBSS to a final concentration of 10-25mM HEPES. |
| |
| Background Fluorescence | Cell sample autofluorescence can reduce fluorescence sensitivity by increasing background 'noise'. Pyridine and flavin nucleotides are intracellular co-enzymes associated with increased cellular autofluorescence, respectively UV-excited blue and Blue-excited green fluorescence. The prescence of protein, unbound antibody and Phenol red will also increase background fluorescence. Although the signal level can be corrected this will result in reduced fluorescent sensitivity. To reduce the effect of background fluorescence wash cells and avoid phenol red. Further, as cells exhibit less autofluorescence when excited by a red laser use red-excitable fluorophores(APC, Alexa647, Alexa700, APC-Cy7 or APC-Alexa750) when autofluorescence is an issue. |
| Vital
Staining (Top) |
|
| Viability | FSC/SSC scatter profiles can be used to approximately discriminate live/dead populations in cell preparations, however when requiring high purity sorts it is recommended that a viability dye is used whenever possible. Propidium Iodide(ex 488nm/em 615nm), 7-Amino-Actinomycin D (ex 488nm/em 660nm) or DAPI(ex 355/407nm /em 455nm) can be used @ ~1ug/ml to excluded cells with compromised membrane integrity. For intracellular stained cells LIVE/DEAD fixable dead cell stain kits from invitrogen come in various colours. |
| Filtration (Top) |
|
| Sample Filtration | To reduce the probability of a blockage in the flow cell nozzle it is recommended that cell suspensions or pre-filtered through a sterile 40-50um mesh filter before sorting eg Partec CellTrics filters |
| Cell Density (Top) |
|
| Cell Density | For cell sorting aim to have cell
suspensions
@ 5-50x106 cells/ml. Remember to bring extra sample medium in case dilution required. Optimal threshold rate for standard sorting is ~22,500 events/sec |
| Adherent
cells (Top) |
|
| Adherent Cells | Adherent cells may cause problems
for sorting. Cells dissociated with trypsin are often neutralised by
the addition of serum. However, reintroduction of divalent cations can
allow the cells to adher to the platicware or other
cells. Always transfer such cells to
polypropylene platicware for cell sorting and replacing serum with a
trypsin inhibitor may be more suitible for sorting purposes. Slower acquisition rates are recommended for adherent cells, activated cells, embryonic stem cells and dendritic cells but this will increase sort period. |
| Aggregation (Top) |
|
| DNase | Cell
aggregation may occur due to the release of DNA in samples with low
viability. Free DNA in the sample media causes cell clumping which
reduces the potential sort efficiency. Aggregates may also block the
sort nozzle leading to premature sort termination! This can be
mitigated by supplementation of the sample media with 10 Units of
DNase/ml in the presence of 1-5mM magnesium chloride. This is highly recommended for adherent cells. |
| Culture (Top) | |
| Secondary Culture | As a precaution add antibiotics to cell recovery media @ 50-100U penicillin plus 50-100ug streptomycin. Most commonly achieved by diluting stock 1:100 in media. |
| Plasticware (Top) |
|
| Sample tubes | Polypropylene tubes are
recommended for sorting due to low protein binding. Either 5ml 12*75mm tubes(eg BD Falcon: 352063) or 15ml conical tubes(eg BD Falcon: 352097) are compatible with the FACSAria. |
| Recovery (Top) | |
| Recovery | 15ml(6 million cells) or 5ml(2 million cells) polypropylene tubes are recommended for standard recovery. Pre-loaded with appropriate media*.(*contact facility) However, cells can be sorted into microtiter plates, tissue culture plates, Petri dishes or directly onto microscope slides. Cells will be damaged by the sorting process - better initial viability/better final recovery. |
| Controls (Top) |
|
| Opitmal Gating | To assist in defining optimal gating for cell sorting the following controls are recommended:
|
| Questions? (Top) |
|
| Problems | E-mail queries not answered here to IIIRFlow |
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