American Family Children's Hospital
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Equipment

Introduction

Training

Analysis Computers

Benchtop Analyzers

Sorters

Microscopes

 

Introduction

Please review Flow Cytometry - Using the Facility before making an appointment. Use iLab to schedule appointments for all Flow Lab equipment.

 

New users must attend the Introduction to Flow Cytometry lecture series before signing up for any training on any cytometer. (Training on the microscope does not require the lecture series.)

 

After attending the lecture series, make an appointment in iLab appointment for two hours on the instrument(s) to be used.

 

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Training

The two-hour hands-on training is intended for those with little or no flow cytometry experience. It will teach the basics of instrument set-up and data collection. Fluorescent Beads will be used for training. Please do not bring cell samples.

 

Training appointments are available at the following times:

  • BD FACSCalibur (7016 WIMR) - Tuesday 12-3pm 
  • BD LSRII - Wednesday 12-3pm
  • BD LSR Fortessa - Thursdays, 12-3pm
  • MACSQuant10 - Thursdays, 12-3pm
  • ImageStream - Monday 12-3pm) 

Once the two-hour training is completed or for those who have some experience operating a flow cytometer, it will be necessary to schedule an hour of assisted time with user samples to discuss specific assay data collection. No user will be allowed to operate the cytometers independently until these training sessions are completed.

 

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Analysis Computers

Offline data analysis computers are available to laboratory users only and are located at both the 7th floor WIMR location and at the McArdle Laboratory. Computers are loaded with a variety of analysis packages including FlowJo, CellQuestPro, FCS Express, ModFit, WinList, and Image analysis, as well as standard Microsoft Office software.

There is no charge for the use of these computers. However, they must be used exclusively for the analysis of data generated at this laboratory. Computers are available on a first-come, first-served walkup basis for users of the facility. 

 

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Benchtop Analyzers

The benchtop instruments are the FACSCalibur, LSRII, Fortessa, and MACSQuant 10, and ImageStream. Use iLab to make appointments.
 
When signing up for appoitments, insure all fluorochromes to be used can be excited with the lasers listed.
 
Before usin gan instrument for the first time, schedule a two-hour training appointment. New users with little flow cystometry experience must first attend the Introduction to Flow Cytometry lecture series.
 
The instruments are available for self-run appointments for which no assistance is provided or for staff-assisted appointments for those who need assistance in data collection or instrument operation. Staff-assisted appointments can be used by those who have no received any instrument training but need samples analyzed, as well as by those who need assay-specific instruction.
 
All users operating cytometers after staff hours (6pm to 8am) or at the satellite facility are responsible fo rshutting down the cytometer after use. Please contact staff with any questions.

 

Instrument Maps:

Use iLab to make an appointment for:

 

BD FACSCalibur (7016 WIMR and 118 McArdle)

The FACSCalibur has two lasers. 488nm has three detectors and 633nm has one detector.


BD LSRII (7016 WIMR)

The LSRII has five lasers. 355nm has three detectors, 405nm has five detectors, 488nm has two detectors, 561nm has five detectors and 642nm has three detectors. Filters can be exchanged for custome configurations.

 

BD LSR Fortessa (7016 WIMR)

The LSRII has five lasers. 355nm has three detectors, 405nm has five detectors, 488nm has two detectors, 561nm has five detectors and 642nm has three detectors. Filters can be exchanged for custome configurations. The Fortessa also has a High-Throughput Sampler for automated sampling of 96 and 384 well plates.

 

MACSQuant10 (7016 WIMR)

The MACSQuant has three lasers. 405nm has two detectors, 488nm has four detectors, and 633nm has two detectors. The instrument is also equipped with tube and plate loaders and an integrated MACS magnetic bead separation column.

 

ImageStream (7016 WIMR)

The ImageStream MarkII is an imaging cytometer for cells in suspension. It has high resolution CCD cameras instead of photomultiplier tubes for detection and is equipped with 20x, 40x and 60x objectives, six detection channels and two lasers (488nm and 642nm). 

 

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Sorters

The facility has two high-speed sorters, both at the 7th floor WIMR location. Each sorter has some unique capabilities, but both can sort up to four separate populations simultaneously or deposit cells into multi-well plates. Operated by staff ONLY. They are both housed under BSL2 conditions. Note: Samples exceeding BSL2 levels when aerosolized cannot be sorted.

 

Sorter Maps:

Use iLab to make appointments on the instruments:


BD FACSAria without Biosafety Cabinet "Jill” 

The SORP BD FACSAria is a digital system equipped with five lasers (UV, 405 nm, 488 nm, 532 nm and 640 nm). The Aria can simultaneously collect fluorescent emissions from 16 different fluorophores.  Please contact Flow Lab staff us if you have questions regarding fluorophore compatibility. 

 

BD FACSAria with Biosafety Cabinet "Jack” 

The FACS AriaIII is a cell sorter equipped with four lasers (403 nm, 488 nm, 561 nm and 637 nm). This sorter is contained in a custom Biological Safety Cabinet. Sorts containing human cells, retroviral transfections or as determined in your lab's Biological Safety Protocol MUST be sorted on this instrument.

 

Adding Flow Cytometry to your Bio ARROW Protocol 

UW Flow Lab Hood Certification Dates

 

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Microscopes

Please consult with the staff before scheduling an appointment if you have not used the microscope before. 

 

Use iLab to make appointments on these instruments:

 

Olympus Microscope 

The Olympus BX51 Fluorescent Microscope is equipped with CCD camera, SPOT imaging software, 10x, 20x and 40x objectives and the following excitation/emission filters:  480/20 ex/510LP em, 535/30 ex/580LP em, 365/10 ex/420LP em, 535/50 ex/346/75 em, and brightfield.

 

Fluorescence Microscope Filter Sets for the Olympus BX51 (pdf)

 

Nexcelom Cellometer

The Nexcelom Cellometer Auto T4 Cell Viability Counter 

 is a PC-based instrument for one-step 4x bright field image capture and analysis. This state-of-the-art cell counter performs count, size, and Trypan Blue viability analyses that are 10x faster and more accurate than manual counting. This instrument is ideal for cell lines and purified primary cells, including MCF-7 breast cancer cells, PC3 prostate cancer cells, and 1600 other cell types, due to its advanced software that assesses clumpy cells and omits debris.

 

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FACSCaliburFirtessaFortessa MACSQuant ImageStream MarkII

AriaIIFACS AriaIII