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Home » Research Cores » Flow Cytometry Core Facility » Sample Submission » Sample Preparation
Sample Preparation

Safety Guidlines

The UAMS flow core is a multi-user facility where many different samples from various sources that contain unknown animal and human pathogens are analyzed and sorted. The safety of the staff and users of the facility is the ultimate concern. Effective immediately the following guidelines will be in effect:

1. No samples BSL 3(or higher) or radioactive samples or any kind will be allowed in the core facility.

2. All samples for analysis must be fixed prior to being submitted for flow cytometry analysis. There are several fixation procedures available on-line and there is also one listed on the flow core website.

3. Unfixed samples may be submitted for sorting but must be BSL1 level only. No BSL 2 or human primary cells will be sorted.

You will be required to provide a signature upon submitting samples that these guidelines have been met and notify personnel of any safety concerns associated with the samples. Failure to follow these procedures will result in no longer being able to use the core facility.

 

Analysis- Fortessa

In order to be analyzed on the Fortessa, samples must be in 5ml- 12x75mm polystyrene round bottom tubes BD Falcon brand. (catalog # 352052, you can order from Fisher) No other tubes will work on the Fortessa.

Samples must be free of aggregates, because this will clog the instrument. If there are clumps you can see, then you will need to filter your samples because they will not be able to be run on the instrument.

You must bring a negative control as well as individual positive controls for each fluorochrome you are using. For example, if you are using FITC and PE for your experiment, you will need to bring your samples as well as a negative control (cells only), and a positive FITC control, and a positive PE control. Samples cannot be properly ran or analyzed without these controls!!

The volume for your samples needs to be at least 0.5 ml. The recommended concentration is 1 x 10 6 cells per ml. The more cells you have, the quicker your samples will run so if you cannot get this concentration, your samples will take longer to run. Please let me know this when you make your appointment so we can schedule more time. The minimum amount of cells we could attempt to run would be about 50,000.

If your samples are not properly prepared or filtered and cause instrument clogging, we will filter your samples for you here and you will be charged our cost for the filter of $1 per filter.

Sorting & Analysis- FACS Aria

Samples can be prepared in either 5ml-12x75 mm polystyrene round bottom tubes BD Falcon brand (catalog # 352052) or 15 ml conical tubes, for the FACS Aria.

Samples ran on the FACS Aria MUST be filtered before running on the FACSAria.  The nozzles are easily clogged by cell aggregates. There are recommended filter cap tubes for use with the Aria. (BD Falcon catalog #352235). If do not have these available for use, we can filter your samples here and I will charge you for the cost of the filters.

You must bring a negative control as well as individual positive controls for each fluorochrome you are using. For example, if you are using FITC and PE for your experiment, you will need to bring your samples as well as a negative control (cells only), and a positive FITC control, and a positive PE control. Samples cannot be properly ran or analyzed without these controls!!

Sorting

Prepare the samples as you would for analysis, including controls, filtering, etc. However, the recommended concentration for sorting is 10 x 10 6 cells per ml; adjust volume accordingly for more or less cells. We can sort as many cells as time allows. On average, it takes about 45 min to set-up and sort 10 million cells.

You must bring collection tubes that we will sort your cells into. Polypropylene tubes are recommended for collection from the FACSAria.  The Aria charges the stream to sort cells and a charge may build around polystyrene tubes as the cells are sorted.  Initial samples may be in either polystyrene tubes or polypropylene tubes. 

When using tubes for collection, it is a good idea to coat them with BSA, especially the 15 ml conical tubes.  This will help keep the cell droplets from sticking to the sides of the tube and drying out.  To coat the tubes, fill them with 4% BSA (bovine serum albumin) in 1X PBS (filter-sterilized) and incubate at 4 o C for at least 1 hour prior to sorting.  Just prior to bringing the tubes for sorting, pour out the BSA (this may be reused for up to 1 month), and then add about 0.5 ml of media to the tubes.

If sorting 1-2 populations, cells can be collected into microtubes, 5 ml 12 x 75 mm tubes, or 15 mL conical tubes

If sorting 3 or 4 populations, cells can be collected into microtubes or 5 ml 12 x 75 mm tubes

We are also able to sort cells into 6-, 24-, 48-, and 96 well plates as well as onto a glass slide.

Since sorting can take an hour or more, it is recommended if you cells need to be kept cold that you bring you samples to the lab on ice.