Flow Cytometry - In House Information
Staff
Adele Barnard
Geza Paukovic
Responsibilities
- Maintanence of FACS machines,
- Teach and help students to use and not abuse the machines,
- All Cell sorting.
- Help implement and advise on different cytometry techniques.
Hours:
9.00 am -5.00 pm, weekdays
Please be aware that we do not get paid for any overtime, so if you want my help then be ORGANISED!
What's in the FACS Room?
FACScan — Detect up to 3 fluorochromes
Advance booking sheets located in FACS room, current week bookings on board out side of FACS room door.
FACSCalibur — Detect up to 4 fluorochromes
Advance booking sheets located in FACS room, current week bookings on board located on the back of FACS room door.
The FACScan and FACScalibur are experimenter operated
FACSsorter — Two way cell sorting or 96 well plate cell sorting
Detect up to 4 fluorochromes
Classified as a dedicated operator machine.
Booking diary located on top of FACSsorter, can also book using Netscape Calendar
When planning your sorting experiment take into account that the machine can take approximately 1-2 hours to be ready and 1 hour to clean the machine after the sort.
Please note that sorting should be regarded like any other specialised technique, and should be acknowledged as such.
Coulter counter
Fluorochromes Commonly Used
- FL1 -FITC or Alexa 488
- FL2 -PE
- FL3 -Tricolour, PerCp, PI, Cychrome C
- FL4 -APC
Sheath Fluid & Waste
- The sheath fluid (saline) is generally made up ready to use in the FACS room.
- To empty the waste container, simply tip the contents down the sink. To the empty container added 100mls of hypochlorite. Be aware that human samples are run on the machines, so it is good to always wear gloves when handling the waste container and a gown when handling the hypochlorite as it will ruin your clothes if split. Unfortunately no clothing allowances come with this job!!!!!
People who run human samples should always empty the waste container when finished acquiring.
Storage of FACS Data
All FACS users are in charge of their own files
After acquisition copy your files IMMEDIATELY onto a Mac-formatted zip disk. When the zip disk is full, the data can be burned onto CD. It might be wise to do 2 CDs, so that one can be at work, the other at home, in case of a fire. Your files can stay on the FACS MACS until analysed or until room is needed. Be warned that if the hard disks are full, FACSfiles over 1 month old will be deleted at random, so make sure you ALWAYS copy to zip disk IMMEDIATELY after sample acquisition.
Analysis of Data
Most, if not all, of the Macintoshes within the department now have the Cell Quest program installed. This means you can analyse your data anywhere, at anytime.
Of course if the FACS machines are not required for acquisition, these can also be used for analysis. However, acquisition takes preference over analysis. In general the machines are usually free in the morning.
Running of Human or Infectious Samples
For all human or infectious samples the following must occur:
- Cover keyboard with cling wrap
- Run hypochlorite followed by water through the machine after acquisition
- Wipe down mouse and keyboard with 70% ETOH after acquisition
- And if highly infectious, empty waste container after acquisition.
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