Sample Preparation

Analysis Cell Preparation

Samples must be in the single-cell suspension. Cells must be filtered (<80um)  at the FACS instrument, even if samples were filtered earlier in the day at the lab bench.

 

The sample must be in a 12 x 75mm Polystyrene tube (Falcon #s 352052, 352054, or 352058). Transfer of samples may be required if using inappropriate tubes.

Minimum volume of each sample is 0.3ml.

Samples containing potential bio-hazards are recommended to be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition. If the user has to run those samples without fixation, he/she must follow the special “Guidelines for running potential bio-hazardous specimen without fixation on STI Fortessa in BST Room S756”

Users cannot discard tubes with cells in fixative solution (e.g. paraformaldehyde) in the Core. All tubes with unfixed human, non-human primate cells or virally-transduced or infected cells must be disposed in your own laboratory by following EH&S recommendations.

No radioactive materials are allowed in the facility.

Running potential bio-hazardous specimen without fixation on STI Fortessa in BST Rm S756

It has been observed that the aerosol can be generated when taking the sample tube off from the sample injection port (SIP).  Since some users may run potential bio-hazardous specimen without fixation, for the purpose of protecting operator as well as other users, this shared instrument is now located inside a class II biosafety cabinet in a Biosafety Level 2 (BSL2) certified room. By following the BSL2 requirements from EH&S, we request that samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition.  If a user has to run a potentially bio-hazardous specimen without fixation, such as unfixed human, non-human primate cells, or virally-transduced or infected cells, in addition to following our general guidelines for using LSR II and Fortessa, he/she must also do the following:

    1. Before starting the experiment, users need to TURN ON the hood

    2. After finishing the experiment, the user MUST decontaminate the working area by spraying Viraguard around and under the SIP areas and let it stay for at least 10 min.  Then follow the “cleaning and shut down procedure” by running 10% Bleach tube for 10 min instead of 5 min.  Before the user leaves the lab, he/she must wipe the areas that have been sprayed with Viraguard by using a paper towel, and then he/she must TURN OFF the hood.

 

Sorter Cell Preparation

Cells (if <40 μm) in all samples and controls be filtered through a <40 μm nylon mesh filter to prevent clogging of the instrument right before they are brought into the sorting room or right before they are introduced into the cell sorter even if the cells have been filtered earlier before staining in the user’s lab.

Samples can be in any of the following tubes: 12 x 75mm, 15ml, or 1ml.

The concentration of lymphocyte-like cells can be up to 5 x 107 cells/ml. The first time user always brings extra sample solution in case it is needed for sample dilution.

For a multicolor experiment, the user must bring an unstained control tube and a single-color stained tube (ideally >5% positive) if the compensation is required for the “color” used.  The user should consult Core staff for the controls required for the instrument set up if there is confusion.

No radioactive materials are allowed in the facility.

Unfixed samples known to contain BSL-3 pathogens or unfixed samples recommended to sort at BSL3 will not be accepted. The Operational Director will make the decision concerning sorting of other known infectious agents on an individual basis in consultation with the EH&S Biosafety Officer when necessary. 

Criteria for samples containing potential bio-hazards, such as unfixed human, non-human primate, virally-transduced or infected cells:

   1. Sample must be contained in a leak-proof container with a secure lid and be clearly labeled with a sample identifier and a biohazard symbol, if needed.

   2. Sample must be clump free and in an appropriate tube for sorting (12 x 75 snap cap tube or 15 ml tube with cap).

   3. Sample must not contaminate the outside of the tube.

Collection Container

Users need to bring a collection container which can be any of the following options:

    1. Tubes: 12 x 75mm, 15ml, or 1ml.  For 4-way sorting, only 12 x 75mm tubes can be used.

    2. Plates: 24-, 96- (including PCR plate), 384-well, or custom size

    3. Trays/dishes or slides

The collection container should be filled with the desired volume of collection medium.

Please comment on any other special handling requirements for your samples, i.e. light sensitivity, tendency to clump, keep on ice, etc. The more we know about your experiment, the better the results.

 

ImageStream Sample Preparation

 

Samples must be in the single-cell suspension. If the user experiences the significant sample clumping, cells need to be filtered through 80um (or less) Nylon mesh (Small Parts, Inc. cat# CMN-0074). 

For manual acquisition, the sample must be in a 1.5ml microcentrifuge tube (siliconized, Sigma, cat# T4816).  For auto-sampler, the samples must be in the round-bottom 96-well plate (Corning, cat#3790). The pierceable cover (X-Pierce, cat# XP-100) is recommended for the plate.  

Cells are recommended to suspend into 1 x PBS with no more than 2% FBS at the concentration of 1 million/50ul.  Minimum volume of each sample is 20ul.

 

Samples containing known biohazard or infectious agents which are ABOVE BSL2 MUST be fixed in a 0.5-2% paraformaldehyde solution for at least 30 minutes before acquisition.  If a user has to run a potentially bio-hazardous specimen without fixation, such as unfixed human, non-human primate cells, or virally-transduced or infected cells, he/she must follow the BSL2 requirements from EH&S.  In addition to that, the user is required to clean the system after finishing the experiment as follows:

    1.) Load and run a tube of 100% bleach (~200ul) for 10 minutes.

   2.) Run a tube of 1X PBS for 5 minutes.