The Flow Cytometry Core (FCC) at MMRI provides instrumentation and expertise in a broad range of basic and medical science disciplines. Samples are prepared by individual investigators, who then deliver samples to the core for flow cytometric analysis or cell sorting. The core has one sorter (BD FACS Aria Fusion) and one analytical cytometer (BD FACS Symphony A3).
The MMRI Flow Cytometry Core Facility offers the following instruments and capabilities:
BD FACS Symphony A3
Sample Preparation: The minimum recommended volume is 500 µl at a concentration of 1×106 cells per ml and cell suspensions must be placed in polystyrene 12x75mm test tubes after filtering them through 45µm filter.
The BD FACS Aria Fusion is capable of high speed and single cell sorting on up to thirteen parameters (forward & side scatter and 11 simultaneous fluorescence colors). It is equipped with four lasers: 405nm, 488nm, 561nm, and 640nm. The BD FACSAria Fusion has the capability of four-way sorting into tubes or single cell sorting directly into plates or slides
This machine has 4 different nozzles: 70 µm, 85 µm, 100 µm and 130 µm in diameter. It is strongly advised to choose a nozzle size that is 3-5 times the diameter of the cells
Sort Sample Requirements:
The outcome of the cell sorting process depends on sample preparation. Most importantly, it is critical to minimize clumps in the sample to be sorted. Clumping is usually caused by inadequate dissociation of cells and/or cell death and release of DNA from dead cells. For these reasons, we recommend the use of a proper sort buffer containing DNAse, and passing the sample through a hypodermic needle prior to sorting
Recommended sort buffer composition:
- 1x PBS or 1x HBSS (Ca/Mg++ free)
- 2mM EDTA
- 25mM HEPES pH 7.0
- 1-5% FCS/FBS (Heat-Inactivated)
- 10units/mL DNase II
- 0.2um filter sterilize
Ideally, cell concentration should not to exceed 106 cells/mL. Cells are to be filtered through 45 uM mesh just prior to sorting (after final wash or staining step). It is also recommended that you bring your samples inside a syringe fitted with a 25 g needle, especially if sample volumes exceed 500 µl; passing samples through the needle prior to sorting will minimize clumping.
Phenol red is not recommended in cell suspension media
Controls for experiments:
An unstained control and a single positive control for each fluorophore used is required
Controls should be brought in a volume of 0.5-1mL and preferably contain approximately 106 cells
Collection Tube Requirements:
It is recommended to collect sorted cells into 12×75 mm GLASS tubes pre- coated with serum/media. Pre-coating collection tubes with serum/media will increase post-sort viability and recovery, while glass minimizes static deflection of charged sort droplets. Sorting into polystyrene and polypropylene tubes may reduce recovery as cells have a tendency to stick to plastic, and these plastics can also build up a static charge All collection tubes should also be filled approximately ¼ with desired buffer or media. It is also recommended to add antibiotics (1x PSN and/or 50ug/ml gentamicin) are added to culture media for samples sorted for cell culture, especially primary cell culture, to prevent contamination.