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Sample Preparation

Cell Sorting:

Set up etc. should be discussed with the facility beforehand; including potential biohazards.

Fill out New Sort Form (xls, 13KB) and email to barry.moran@tcd.ie.

Cell preparation and staining should be optimised, and antibodies titrated.
Staining should be done in a max of 3 x 10⁷ cells/ml. Some general points:

1. When setting up samples for sorting, cells should be:

• filtered pre-sort to reduce clumps and aggregates. Filters are available in 50µM and 30µM sizes from the facility.
• prepared in a sterile environment into a sterile Falcon FACS tube (available from facility), and kept on ice/in dark etc. as appropriate.
• at a concentration up to ~5 x 10⁷/ml.
• ideally in a volume ≥ 0.4ml and ≤ 1ml.
• with a viability marker. Propidium Iodide (PI) is available in the facility.
• in a suitable buffer to ensure optimal cell viability.

Culture media may not be an ideal sort buffer for two reasons: the pH regulation fails under normal atmosphere causing the media to become basic; and the calcium chloride in most culture media is not compatible with the phosphate component of the sheath fluid leading to precipitation of calcium phosphate crystals.

2. In addition, with every sort, please supply:

• suitable buffer/media to collect the cells (e.g. PBS + 1% H-I FBS). Though the sorter is housed in a biosafety cabinet, antibiotics may be advisable. A great reference is TSRI resource
• negative controls; - a small preparation (~0.5-1 x10⁵ in ≥ 0.3ml) of unstained cells.
• compensation controls (if more than one fluorochrome used); - a small preparation of sample (cells or beads) with a single stain for each fluorochrome used (with an identifiable neg and pos population).
• FMO/positive/isotype controls as appropriate.

Cell Analysis:

Sample preparation and experimental design can be decided by the user, with training and/or assistance provided as required.

In general:

• ensure single cell suspension with no clumps as these will block the cytometer. Filters available in the facility.
• cells to be analysed should be in a volume >300µl.
• cells at a concentration between 1 x10⁵ - 2 x10⁷ per ml are adequate, although unless very high acquisition rates are required, 2 x10⁵ - 2 x10⁶/ml are usually optimal.
• a negative, unstained, sample (~0.5-1 x10⁵ cells in ≥ 0.3ml) should be acquired.
• compensation controls (if more than one fluorochrome used); - a small preparation of sample (cells or beads) with a single stain for each fluorochrome used (with an identifiable neg and pos population).
• FMO/ positive/ isotype controls as appropriate; FMOs are ideal to help identify and gate specific populations, and to control for spectral overlap between fluorescences.