Instrumentation

Flow cytometry instruments are located in West 362-364; Imaging cytometry (Amnis ImageStreamx) is located in West 480.

Cell Sorting Instruments

Analysis Instruments

Imaging Cytometry Instrument

FACSAriaII

LSRII 14-Color

Amnis ImageStreamx

MoFlo AstriosEQ

LSRII 18-Color

MoFlo Legacy

FACSCalibur

 

Sorting Instruments

FACSAriaII 16-color cell sorter, BD (purchased 2009, Violet laser upgrade 2013)

● Operated by facility personnel only
● Four lasers; Blue (488nM), Red (640nM), Violet (405nM), UV (355nM)
● Capable of sorting up to 4 subpopulations simultaneously at rates up to 21,000 cells/second (70u) or 9,000 cells/sec (100u)

Suggested fluorochromes (there are many more – see spectra viewers):

Blue Laser (488nM) x 5 parameters

FITC / GFP / Alexa488 (530/30)

PE / DsRED / PyroninY (575/25)

PE-TxRED / PI / mCherry (610/20)

PerCP-Cy5.5 / PE-Cy5 or 5.5 / 7AAD (710/50)

PE-Cy7 (780/60)

Red Laser (640nM) x 3 parameters

APC / Alexa647 (670/30)

Alexa 700 (710/50)

APC-Cy7 / Alexa 750 or 780 (780/60)

Violet Laser (405nM) x 6 parameters

PacBlue/BV 421/CellTrace Violet (450/50)

AmCyan/Aqua Live-Dead/BV 510 (510/50)

BV 570/Qdot 585 (585/30)

BV 605/Qdot 605 (610/20)

BV 650/Qdot 655 (660/20)

BV 685/Qdot 705 (710/50)

UV Laser (325nM) x 2 parameters

Hoechst Red / Qdot 655 (670/50)

Hoechst Blue / DAPI / BV 421 (450/50)

Numbers in parentheses refer to emission filter wavelengths of each detector

ARIA II configuration diagram

Contact facility with questions regarding color combinations.

 

MoFlo AstriosEQ, 32 parameter cell sorter, Beckman Coulter (purchased 2014)

●Operated by facility personnel only
●Seven lasers; UV (355nM), Violet (405nM), Blue (488nM), Green (532nM), Green/Yellow (561nM), Orange (592nM), Red (640nM)
●Capable of sorting to 6 subpopulations simultaneously at rates up to 30,000 cells/second (70u) or 10,000 cells/sec (100u)
●Capable of sorting defined numbers onto plates up to 1536 wells

Suggested fluorochromes (there are many more – see spectra viewers):

UV Laser (355 nM) x 3 parameters

Hoechst Blue / DAPI / BV 421 (458/59)

BV 605 / Zombie Yellow (620/29)

Hoechst Red / BV 650 (692/75)

Violet Laser (405 nM) x 7 parameters

BV421 / PacBlue (448/59)

BV510 / AmCyan / Aqua Live/Dead (513/26)

BV570 / Zombie Yellow (576/21)

BV605 / Zombie Yellow (614/20)

BV650 (664/20)

BV711 (710/45)

BV785 (795/70)

Blue Laser (488 nM) x 7 parameters

SSC (488/6)

FITC / GFP / Alexa488 / CFSE (513/26)

PE (576/21)

Pe-TxRed / PE-Dazzle594 / PI (622/22)

PE-Cy5 / PerCP (664/22)

PE-Cy5.5 / PerCP-Cy5.5 (710/45)

PE-Cy7 (795/70)

Green Laser (532 nM) x 6 parameters

SSC (530/11)

PE / Pyronin Y / YFP (576/21)

Pe-TxRed / PE-Dazzle594 / PI (622/22)

PE-Cy5 / PerCP /7AAD (664/22)

PE-Cy5.5 / PerCP-Cy5.5 (692/18)

PE-Cy7 (736/47)

Grn/Yellow Laser (561 nM) x 4 parameters

SSC (561/4)

PE / DsRed (579/16)

Pe-TxRed / PE-Dazzle594 / PI (614/20)

PE-Cy5 / PE-Cy5.5 / 7AAD (692/75)

Orange Laser (592 nM) x 1 parameter

mCherry / TxRed / Alexa610 (620/29)

Red Laser (640 nM) x 4 parameters

SSC (642/10)

APC / Alexa647 (671/30)

Alexa700 / APC-Cy5.5 (722/44)

APC-Cy7(H7) / Alexa750 or 780 (795/70)

Numbers in parentheses refer to emission filter wavelengths of each detector

Contact facility for other fluorochromes not listed, or with questions regarding color combinations.

 

MoFlo Legacy 5-color Sorter, DakoCytomation (purchased 2001)

● Operated by facility personnel only
● Two lasers; Blue (488nM), Red (635nM)
● Capable of sorting to 4 subpopulations simultaneously at rates up to 25,000 cells/second (70u) or 10,000 cells/sec (100u)

Suggested fluorochromes (there are many more – see spectra viewers):

Blue Laser (488nM) x 3 parameters

FITC/GFP

PE

PerCP(Cy5.5)/PE-CY5(5.5)/ 7AAD 

Red Laser (635nM) x 2 parameters

APC

Alexa 700 or APC-Cy7/Alexa 750/780

Numbers in parentheses refer to emission filter wavelengths of each detector    

Contact facility for other fluorochromes not listed, or with questions regarding color combinations.

Analysis Instruments

LSRII (14-color), Becton-Dickinson (purchased 2007)

● Capable of user-friendly analysis
● Four lasers; Blue (488nM), Violet (405nM), Red (635nM), Green (532mM)
● High-speed analysis-only instrument

Suggested fluorochromes (there are many more – see spectra viewers):

Blue Laser (488nM) x 3 parameters

SSC

FITC / Alexa488 / GFP / YFP / CSFE (530/30)

PE_Blue (575/26)

PerCP-Cy5.5 / PI / 7AAD (695/40)

Violet Laser (405nM) x 3 parameters

BV510 / AmCyan / Aqua Live/Dead (525/50)

BV421 / PacBlue / DAPI/ CellTraceViolet (450/50)

BV605 or 655 / Qdot 605 or 655 (670/14 or 610/20)*

Red Laser (635nM) x 3 parameters

APC / Alexa647 (660/20)

Alexa700 / APC-Cy5.5 (710/50)

APC-Cy7 / -H7 / Alexa750 or 780 (780/60)

Green Laser (532nM) x 5 parameters

PE-Gr (585/42)

PE-TxRed / dsRed / mCherry (610/20)

PE-Cy5 / PerCP (670/30)

PE-Cy5.5 / PerCP-CY5.5 (730/45)

PE-Cy7 (780/60)

Numbers in parentheses refer to emission filter wavelengths of each detector

*Contact facility personnel for instructions

LSR14 configuration diagram

Contact facility with questions regarding color combinations.

 

LSRII (18-color), Becton-Dickinson (purchased 2008)

●Capable of user-friendly analysis
●Four lasers; Blue (488nM), Violet (405nM), Red (640nM), Green (532mM)
●High-speed analysis-only instrument

Suggested fluorochromes (there are many more – see spectra viewers):

Blue Laser (488nM) x 3 parameters

SSC

FITC / Alexa488 / GFP / YFP / CSFE (525/50)

PerCP-Cy5.5 / PI / 7AAD (710/50)

Violet Laser (405nM) x 8 parameters

BV421 / PacBlue / CellTraceViolet / eFluor (450/50)

BV510 / AmCyan / Aqua Live/Dead (525/50)

BV570 / Qdot 565 (560/40)

Qdot585 (585/42)

BV605 / Qdot 605 / ZombieYellow (605/40)

BV650 / Qdot 655 (660/40)

BV711 / Qdot 705 (705/70)

BV785 / Qdot 800 (780/60)

Red Laser (640nM) x 3 parameters

APC / Alexa647 (660/20)

Alexa700 / APC-Cy5.5 (710/50)

APC-Cy7 / -H7 / Alexa750/790/ APC-eFluor780 (780/60)

Green Laser (532nM) x 5 parameters

PE-Gr (575/25)

PE-TxRed / dsRed / mCherry / PI (610/20)

PE-Cy5 / PerCP / 7AAD (660/20)

PE-Cy5.5 (710/50)

PE-Cy7 (780/40)

Numbers in parentheses refer to emission filter wavelengths of each detector

LSR18 configuration diagram

Contact facility with questions regarding color combinations

 

 

FACSCalibur, Becton-Dickinson (purchased 2004)

●Capable of user-friendly analysis
●Two lasers; Blue (488nM), Red (635nM)
●Low-speed (up to 5,000 events per second) analysis-only instrument

Suggested fluorochromes:

Blue Laser (488nM) x 3 parameters

FITC / GFP / Alexa488 / GFP / YFP

PE / DsRED

PE-TxRED / PI or 7AAD / mCherry /

PerCP-Cy5.5 / PE-Cy5 or 5.5 / PE-Cy7

Red Laser (635nM) x 1 parameter

APC / Alexa647 / Alexa 700 or 750 or 780

Contact facility for other fluorochromes not listed, or with questions regarding color combinations.

Imaging Cytometry Instrument

Amnis ImageStreamx Imaging Flow Cytometry, Amnis (purchased 2010)

●Operated by facility personnel only at this time
●1 camera, 6 channels:  Brightfield +  Four lasers; Violet (405nM), Blue (488nM), Red (658nM), IR (785nM)
●IDEAS post-acquisition analysis software (PC) available on dedicated workstation, or may be downloaded by investigator

Suggested fluorochromes:


   

Excitation Laser (nm)

  
ChannelBand (nm)405488658785
  DAPI, Hoechst, PacBlue,   
1430-505Alexa405, DyLight405, CFP,   
  Live/Dead Violet   
  PacOrange, CascadeYellow,FITC, Alexa488, GFP, YFP,  
2480-560Alexa430, QDot525DyLight488, PKH67,  
   MitoTrackerGreen  
   PE, PKH26, Cy3, Alexa555  
3560-595QDot565, QDot585DsRed  
      
   PE-TxRed, ECD, 7AAD,  
4595-660QDot625, eFluor625PI, RFP, mCherry, QDot625  
      
   PE-Cy5, Pe-A647, PerCP,Alexa647, Alexa660, Alexa680, 
5660-745QDot705 PerCP-Cy5.5, DRAQ5, QDot705DRAQ5, APC, Cy5, DyLight649, 
    DyLight680, PE-Cy5, PerCP(-Cy5.5) 
   Pe-Cy7, PE-Alexa750/780,APC-Cy7, APC-Alexa750/780, 
6745-800QDot800QDot800Cy7, Alexa750, DyLight750,PE-Cy7,SSC
    PE-Alexa750 

Recommended fluorochromes (based on optimal excitation and detection channels) are in boldface

Contact facility for other fluorochromes not listed, or with questions regarding color combinations

 

 

Other related equipment available

                Fluorescent microscope

                Additional PC and Mac workstations for post-acquisition analysis

                FlowJo post-acquisition software

                Refrigerator

There is also a collection of the following publications and resources:

Current Protocols in Cytometry

Cytometry, The Journal of the International Society for Analytical Cytology (1988 – current)

● Various flow cytometry reference books, including Practical Flow Cytometry (Shapiro, both 2nd & 3rd editions), Handbook of Flow Cytometry Methods (Robinson), and more

● Catalogs of flow cytometry-related supplies, antibodies, fluorochromes, etc.