The Flow Cytometry Core Facility is equipped with 6 flow cytometers that are categorized into 2 types:

• Bench top analyzer flow cytometers that are operated by the investigators themselves and
• High-speed cell sorter flow cytometers that are operated by the core facility's personnel as a service to investigators only.

The flow cytometers in the facility are equipped with an amazing variety of laser excitation wavelengths, which are shown in the following table:

Laser Lines in the Flow Cytometry Core Facility at Einstein
	High Speed Cell Sorter Flow Cytometers		Bench Top Analyzer Flow Cytometers				Laser Scanning Cytometer
Laser Line (nm)	MolFlowXDP	FACSAria	Aurora	LSRIIs	DXP 10 Calibur	CantoII	iCys
UV (350)	√			√
Violet (407)	√	√	√	√	√		√
Cyan (457)	√
Blue (488)	√	√	√	√	√	√	√
Green (514-532)	√	√
Yellow (561/568)	√	√	√	√	√
Orange (592)	√
Red (633/647)	√	√	√	√	√	√	√
Far Red (676)	√
Infrared (752)	√

Core Facility Bench Top Analyzer Flow Cytometers:  

There are 5 bench top analyzer flow cytometers in the core. One four laser Aurora with auto sampler from Cytek, two BD LSRIIs, one CantoII from BD Bioscience, and one DXP10 Calibur upgraded by Cytek.

We have recently acquired a Cytek Aurora, new and very powerful flow cytometer, for the Flow Cytometry Core Facility. This new instrument uses a unique technology involving multispectral analysis that gives better sensitivity and resolution than our LSR II instruments. It has the capability to do >20 color analysis and uses a remarkable “unmixing” processing algorithm to make fluorescence compensation incredibly simple. This instrument is online for routine booking now. The cost for use will be similar to the current hourly cost for the LSR II.

This prodigy incorporating a unique combination of patent-pending innovative technologies that takes flow cytometry to the next level of performance and flexibility.

With four lasers( 405, 488, 561 and 640nm), three scattering channels, and 48 fluorescence channels, the Aurora suits every laboratory’s needs, from simple to high complexity applications. A paradigm shifting optical design provides unprecedented flexibility, enabling the use of a wide array of new fluorochrome combinations without reconfiguring your system for each application. The state-of-the-art optics and low-noise electronics provide excellent sensitivity and resolution. Flat-top beam profiles, combined with a uniquely designed fluidics system, translate to outstanding performance at high sample flow rates.

 

Available Dyes in the Corresponding Channels:  

Violet Excited Fluors(405nm)	Peak Channel	Band Pass filter
BV421	V1	428/15
Alexa 405, SuperBright 436	V2	443/15
efluor450, VioBlue, Pacific Blue	V3	458/15
BV480	V4	473/15
eFluor 506	V5	508/20
BV510, VioGreen	V7	543/21
BV570, Pacific Orange	V8	577/20
BV605, SuperBright 600, Qdot 605	V10	615/20
BV650, SuperBright 645, Qdot 655	V11	664/27
BV711, Super Bright 702, Qdot705	V13	720/29
BV750, BB700	V14	750/30
BV785, BV786, Qdot 800	V15	780/30
Blue Excited Fluors (488nm)	Peak Channel	Band Pass filter
Alexa Fluor 488, FITC, vioBright FITC, Vio 515, sVio 515, BB515	B2	524/20
Alexa Fluor 532	B3	543/21
PerCP	B8	678/18
PerCP-Cy5.5	B9	697/19
PerCP-eFluor 710, PerCP-Vio 700	B10	717/20
Yellow Green Excited Fluors (561nm)	Peak Channel	Band Pass filter
PE	YG1	577/20
PE-Dazzle 594, PE-CF 594, PE-TexasRed, PE-efluor 610, PE-Alexa Fluor 610	YG3	615/20
PE-Cy5	YG5	678/18
PE-Cy5.5, PE-AlexaFluor 700	YG7	720/29
PE-Cy7, PE-Vio 770	YG9	780/30
Red Excited Fluors (640nm)	Peak Channel	Band Pass filter
APC	R1	660/17
Alexa647, Vio 667, sVio 667, efluor660	R2	678/18
Alexa 700, APC-R700	R4	717/20
Zombie NIR	R6	760/23
APC-Alexa750, APC/Fire 750, APC-Cy7, APC-Vio 770, APC-efluor780, APC-H7	R7	783/23

Available Detectors:  

Laser	Channel	Center Wavelength (nm)	Bandwidth (nm)	Start (nm)	End (nm)
Violet	V1	427.5	15	420	435
	V2	443	15	436	451
	V3	458	15	451	466
	V4	473	15	466	481
	V5	508	20	498	518
	V6	524.5	17	516	533
	V7	541.5	17	533	550
	V8	580.5	19	571	590
	V9	598	20	588	608
	V10	615	20	605	625
	V11	664	27	651	678
	V12	691.5	28	678	706
	V13	720	29	706	735
	V14	749.5	30	735	765
	V15	779.5	30	765	795
	V16	811.5	34	795	829

Laser	Channel	Center Wavelength (nm)	Bandwidth (nm)	Start (nm)	End (nm)
Blue	B1	508	20	498	518
	B2	524.5	17	516	533
	B3	541.5	17	533	550
	B4	580.5	19	571	590
	B5	598	20	588	608
	B6	615	20	605	625
	B7	660	17	652	669
	B8	678	18	669	687
	B9	697	19	688	707
	B10	717	20	707	727
	B11	738	21	728	749
	B12	760	23	749	772
	B13	783	23	772	795
	B14	811.5	34	795	829

Laser	Channel	Center Wavelength (nm)	Bandwidth (nm)	Start (nm)	End (nm)
Yellow Green	YG1	577	20	567	587
	YG2	598	20	588	608
	YG3	615	20	605	625
	YG4	660	17	652	669
	YG5	678	18	669	687
	YG6	697	19	688	707
	YG7	720	29	706	735
	YG8	749.5	30	735	765
	YG9	779.5	30	765	795
	YG10	811.5	34	795	829

Laser	Channel	Center Wavelength (nm)	Bandwidth (nm)	Start (nm)	End (nm)
Red	R1	660	17	652	669
	R2	678	18	669	687
	R3	697	19	688	707
	R4	717	20	707	727
	R5	738	21	728	749
	R6	760	23	749	772
	R7	783	23	772	795
	R8	811.5	34	795	829

Suggested 23 Dye Combination:  

Violet Excited Fluors	Blue Excited Fluors	Yellow Green Excited Fluors	Red Excited Fluors
BV421	Alexa Fluor 488	PE	APC
SuperBright 436	Alexa Fluor 532	PE/Dazzle594	Alexa647
efluor450	PerCP-Cy5.5	PE-Cy5	APC-R700
BV480	PerCP-eFluor 710	PE-Cy7	APC/Fire750
BV510
BV570
BV605
BV650
BV711
BV750
BV785

Becton Dickinson LSRII-Yellow: The BD™ LSR II flow cytometer is the most flexible yet powerful benchtop analyzer available. Innovative technology in the BD LSR II optics and digital electronics have created a more sensitive flow cytometer that yields more information from each sample.

 

The second LSRII we purchased recently has 5 lasers, 355nm, 405nm, 488nm, 561nm and 640nm. It can analyze up to 14 colors simultaneously.

LSRII Yellow Configuration
Laser	Detector Name	Bandpass Filter	Dichoric Filter	Fluorochrome Detected
Blue Laser (488 nm)	A	710/50	685 DLP	PerCP-Cy5.5, PerCP
	B	575/26	550 DLP	PI
	C	525/50	505 DLP	FITC, GFP, YFP
	D	488/10	Blank	SSC
Red Laser (640 nm)	A	780/60	750 DLP	APC-Cy7
	B	730/45	685 DLP	Alex700
	C	660/20	Blank	APC, Alex647
Yellow Laser (561 nm)	A	780/60	750 DLP	PE-Cy7
	B	670/30	645 DLP	PE-Cy5
	C	610/20	600 DLP	PE-TxRed-YG, Mcherry
	D	582/15	Blank	PE-YG, RFP
violet Laser (405 nm)	A	525/50	505 DLP	Am Cyan
	B	450/50	Blank	Pacific Blue
UV Laser (355 nm)	A	530/30	505 DLP	Indo-1 (blue)
	B	450/50	Blank	DAPI, Indo-1 (Violet)

Becton Dickinson LSRII-U: We recently add a 561nm Yellow laser to this LSRII.

 

The LSRII-U has optical detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the FACSCalibur. The core's LSRII-U has 5 lasers with 2 detectors for laser-light scatter and 13 detectors for fluorescence in the following configuration:

• a 20 milliwatt solid-state argon laser that emits blue light at 488 nanometers and which is connected to a unique Trigon optical detector 'block' that's equipped with 3 detectors (2 fluorescence and 1 laser-light scatter), which are detailed below
• a 40 milliwatt solid-state laser that emits red light at ~640 nanometers and is connected to a unique trigon optical detector block that's equipped with 3 fluorescence detectors
• a 20 milliwatt laser solid-state UV that emits non-visible ultra-violet light at 350 nanometers and which is also connected to trigon optical detector block that's equipped with 2 fluorescence detectors
• a 100 milliwatt solid-state violet laser that emits violet light at 407 nanometers and which is connected to trigon optical detector block that's equipped with 2 fluorescence detectors
• a 50 milliwatt solid-state yellow laser that emits red light at ~561 nanometers and is connected to a unique Octagon optical detector block that's equipped with 4 fluorescence detectors

LSRII-U optical configuration:  

 

LSRII-U Configuration
Laser	Detector Name	Bandpass Filter	Dichoric Filter	Fluorochrome Detected
Blue Laser (488 nm)	A	695/40	685 DLP	PerCP-Cy5.5, PerCP
	B	530/30	505 DLP	FITC, GFP, YFP
	C	488/10	Blank	SSC
Red Laser (640 nm)	A	780/60	735 DLP	APC-Cy7
	B	710/20	685 DLP	Alex700
	C	670/30	Blank	APC, Alex647
Yellow Laser (561 nm)	A	780/60	735 DLP	PE-Cy7
	B	670/14	635 DLP	PE-Cy5
	C	610/20	600 DLP	PE-TxRed-YG, Mcherry
	D	586/15	Blank	PE-YG, RFP
violet Laser (405 nm)	A	525/50	505 DLP	Am Cyan
	B	440/40	Blank	Pacific Blue
UV Laser (355 nm)	A	530/30	505 DLP	Indo-1 (blue)
	B	440/40	Blank	DAPI, Indo-1 (Violet)

CyFlow Cube6: is a Portable, Dual-Laser Flow Cytometry System

 

CyFlow® Cube 6 is ultra-compact and uniquely designed for all applications in cell analysis and absolute counting. It employs 6 optical parameters. Forward Scatter (FSC) and Side Scatter (SSC) work in combination with 4 fluorescence channels (FL1-FL4).

Highlights:

• Absolute cell counting without counting beads
• Superior nanotechnology: small particle detection < 50 nm
• Optional CyFlow® Robby autoloader for well plates and tubes

CyFlow Cube6 Detector Configuration
Laser	Detector Name	Bandpass Filter	Dichoric Filter	Fluorochrome Detected
Blue Laser (488 nm)	SSC	488/10	Blank	SSC
	FL1	530/40	500 DLP	FITC, GFP, YFP
	FL2	580/50	560 DLP	PI
	FL3	675/20	620 DLP	PerCP-Cy5.5, PerCP
Red Laser (638 nm)	FL4	650 LP	Full Mirror	APC, Alex647, Alex700APC-Cy7

Becton Dickinson FACSCalibur: DxP10 FACSCalibur upgrade for our FACSCalibur make the cytometer capable of 10 channels of fluorescent detection excited by four lasers (488/561/637/407). Electronic signal processing is also upgraded from the original analog paths to full digital processing. Acquisition control has the added benefit of the long trusted analysis software, FlowJo, in a "Collectors" edition suite.

 

As a summary, the upgrade that includes the items mentioned above will result in the following final instrument configuration:

• 4-laser system (488/561/637/407nm); 4 detection channels off of the 488 laser, 2 detections channels each off of the 561, 637 and 407 lasers; FSC, SSC and 1 selectable Area and Width channel; 18 bit, 4.5 log decade digital data, data transfer rate of 48Mhz, system clock speed 4Mhz, ADC clock speed 500Khz; Real time spillover (compensation) matrix; View data with auto-calculated compensation during live acquisition; Data file will contain raw and compensated parameters; User definable spillover values.

DXP10 FACSCalibur Configuration
Laser	Detector Name	Bandpass Filter	Dichoric Filter	Fluorochrome Detected
Blue Laser (488 nm)	BluFL-1	530/30	560 DSP	FITC, GFP, YFP
	BluFL-2	580/45	640 DSP	PE
	BluFL-3	695/40	725 DSP	PerCP, 7ADD, PerCP-Cy5.5
	BluFL-4	740LP	Blank	PE-Cy7
	SSC	480/10	505 DSP
Red Laser (637 nm)	L2-1	666/27	660 DSP	APC, Alex647
	L2-2	740LP		APC-Cy7
Yellow Laser (561 nm)	L2-3	590/20	600 DSP	RFP, PE
	L2-4	615/25		Mcherry, PE-Texas Red
violet Laser (407 nm)	L3-1	450/50	480 DSP	Alex405, DAPI, PB
	L3-2	545/30		Alex430

Becton Dickinson FACScan analog bench top analyzer flow cytometers [4-color, 2-laser systems]; available to trained users with online booking.

 

The core is equipped with 1 upgraded FACScan flow cytometer that is equipped with a low-power (15mW) argon laser that emits blue light at 488 nanometers as well as a low-power (30mW) helium-neon diode laser that emits red light at 633 nanometers. The core's FACScan is upgraded from the factory's original configuration that has only 1 laser and 3 fluorescent detectors. It has two detectors for laser-light scatter and 4, not 3, detectors for fluorescence in the green, orange and red/dark red regions of the color spectrum. The detector array permits the use of a great number of fluorophores, allowing for up to 7 parameters for every particle interrogated (time is also a parameter). The optical filters on the FACScan are not interchangeable (each fluorescence detector "sees" on a single fluorescence color) so the choices of fluorophores are similarly constrained, however, a tremendous number of fluorophores can be detected with it's configuration.

 

Both FACScans are equipped with pulse processing for cell doublet discrimination and have an optional 20-liter cubitainer system that has 5 times the buffer and waste capacity of the factory system.

Becton Dickinson FACS CantoII: The BD FACSCanto II features many innovations for both clinical and research labs, including a true fixed-alignment flow cell to minimize startup time and improve reproducibility. With High Throughput Sampler (HTS) option available for this flow cytometer. The HTS provides fully automated and rapid sample acquisition from either a 96- or 384-well microtiter plate.

 

FACS CantoII Configuration
Laser	PMT position	BP Filter	LP mirror	Intended Dye
488 nm	A	780/60	735DLP	Pr-Cy7
	B	670LP	655DLP	PerCP, PerCP-Cy5.5
	C	Blank	610DLP	---
	D	585/42	556DLP	PE, PI
	E	530/30	502DLP	FITC, Alex488
633 nm	A	780/60	735DLP	Apc-Cy7, Alex750, Alex780, APC-eFluor 750
	B	Blank	685DLP
	C	660/20		APC, Alex633, Alex647, To-PRO-3

Core Facility High-Speed Cell Sorter Flow Cytometers:  

There are 3 high-speed cell flow cytometers in the core facility that consist of 3 different models by 2 manufacturers. The facility is equipped with a DakoCytomation MoFlo and a Dako (formerly DakoCytomation) MoFlo XDP. Dako is based in Fort Collins, Colorado. The 3^rd sorter in the facility is a Becton Dickinson FACSAria.

The primary function of the high-speed cell sorter flow cytometers are for physically sorting cells or particles of interest and for analysis requirements that cannot be met on the bench top analysis flow cytometers in the facility. For example, the sorter flow cytometers are the only flow cytometers in the facility that are equipped with cyan, green, yellow, orange or far red laser lines, consequently, investigators requiring access to these excitation lines are granted access to the sorter flow cytometers regardless if their goal is to physically sort cells (particles) of interest, otherwise, as stated previously, the primary function of the sorter flow cytometers is for physically sorting cells or particles of interest.

Unlike the bench top analyzer flow cytometers, which are operated by the investigators themselves, sorter operation is provided as a service. This is because the instruments are much more complex than the bench top analyzers and consequently require much more experience and training to achieve proper function. Also, because of the open design of the MoFlos, which permits great flexibility in experimental design, their components are more easily damaged and they are expensive to replace.

Because the sorters use a stream-in-air sorting method, they aerosolize the samples. Consequently, the sorters cannot be used for sorting hazardous samples. Non-hazardous living cells can be sorted and recovered in sterile form for subsequent culture or for in vitro functional studies. Accommodations can be made for AECOM investigators requiring sorting of hazardous specimens off site - please inquire in advance.

DakoCytomation MoFlo high-speed cell sorter flow cytometer [8-color, 3-laser system with optional 4-way sorting, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking.

 

The MoFlo has three high-powered tunable-wavelength lasers which consist of an argon, a krypton and an argon-krypton mixed-gas laser. The MoFlo also has detectors and removable filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the other cytometers, i.e., mCherry, mRFP, Texas Red (yellow laser excitation), among many others. There are other important differences between this cytometer and the others in the facility. Among them are:

• The MoFlo, like all jet-in-air sorters, generally has less sensitivity for immunofluorescence than the bench top analyzer flow cytometers, which use a closed-cuvette for fluorescence detection.
• Sorter flow cytometers such as the MoFlo can physically sort up to four populations concurrently and the MoFlo is able to sort them into a great variety of collection devices:
  
  • Microscope slides,
  • Microtubes,
  • 12 x 75 mm polypropelyne test tubes,
  • 14, 15 or 50 ml polypropelyne tubes,
  • Multiwell plates (24-, 96-, or 384-well plates, etc.; 60- or 72-well Terasaki, etc.),
  • A choice of a variety of custom devices that are only limited by the space constraints within the sort chamber area.
   

The diminished fluorescence detection of the MoFlo, like all jet-in-air sorters, over the bench top analyzers is a property of the fluidic and optical system that is needed for sorting. The three bench top analyzers have a closed cuvette that is optically coupled to the fluorescence collector lens which is analogous to an oil immersion objective on a microscope whereas the MoFlo has a jet-in-air system with a "dry" fluorescence collector lens. Cells or particles and enveloping sheath are pushed out of a small ceramic nozzle as a fine stream that crosses the laser beam(s) and fluorescence is collected by a dry microscope objective that is focused on the laser/stream intersection. It follows that, as with dry microscope objectives, less light is collected, the sensitivity will always be less. The magnitude of the difference in the fluorescence varies with the fluorochrome but a general rule is for the jet-in-air sorters to have approximately a half-decade lower fluorescence than the bench top analyzers. . It is a common misconception that a sorter is able to do everything that a bench top analyzer is able to do plus a whole lot more simply because a sorter costs more than they do.

As mentioned previously, the MoFlo is equipped with three high-powered tunable-wavelength lasers, which are detailed in the following table.

The typical configuration for immunophenotyping using the MoFlo is detailed below:

• The tunable argon laser to be tuned to its blue 488-nanometer line and for that laser to be placed into the primary laser pathway which contains 6 optical detectors (2 optical detectors for laser light scatter and 4 for fluorescence detection),
• The tunable krypton laser to be tuned to its multi-line violet mode (concurrent 407-, 413- and 415-nanometer lasing) and for that laser to be placed into the secondary laser pathway that contains 2 fluorescence detectors and
• The tunable argon-krypton mixed gas laser to be tuned to its red 647-nanometer line and for that laser to be placed into the tertiary laser pathway that also contains 2 optical detectors.

A strength of the open architecture of the MoFlo design is that any of its lasers can be placed into any of its optical pathways.

Persons may be interested to know that the MoFlo was conceived at Lawrence Livermore National Laboratories (home of the world's fastest computer!) as a tool for the rapid isolation of chromosome-specific DNA libraries for the Human Genome Project. The MoFlo was the first commercially available high-speed cell sorter flow cytometer when it was introduced in 1994.

Dako MoFlo XDP high-speed cell sorter flow cytometer [12-color, 4-laser system with optional 4-way sorting, aerosol containment, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking.

 

The MoFlo XDP - AECOM's was the first commercial installation of this model flow cytometer in the world - is almost identical in 'architecture' to the core's 'original' (existing) MoFlo. The 2 MoFlos have the identical optical pathways and fluidic systems though the MoFlo XDP is equipped with 12, not 8, fluorescent detectors. The primary difference between AECOM's 2 MoFlos is in the electronics where the MoFlo XDP uses completely digital processing whereas the original MoFlo uses analog-to-digital processing though there is no difference in their sorting speed. The fluorescent detectors are a newer generation than those that exist in the existing MoFlo and the company claims that the XDP is more sensitive than the original MoFlo. Perhaps the most notable difference between the 2 MoFlos is that the MoFlo XDP is equipped with 4, not 3, lasers and, as mentioned previously, there are 12, not 8, fluorescent detectors.

The MoFlo XDP has 3 high-powered tunable-wavelength lasers, which consist of an argon, a krypton and an argon-krypton mixed-gas laser (virtually identical as the original MoFlo) though, unlike the 'original' MoFlo, the MoFlo XDP is also equipped with a high-powered 592nm (orange) fiber laser, which shares the mixed-gas laser pathway. Investigators that are interested to utilize the orange fiber laser must forgo concurrent use of the mixed-gas laser. The MoFlo XDP's lasers are detailed in the following table.

Like the original MoFlo, the MoFlo XDP has detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the bench top analyzer cytometers, such as mCherry and Texas Red (yellow laser excitation) as well as the newly published far-red fluorescent proteins mKate and Katushka (orange laser excitation), among many other fluorophores. As with the original MoFlo, there are other important differences between this cytometer and the bench top analyzer flow cytometers in the facility. Among them are:

• The MoFlo XDP, like all jet-in-air sorters, generally has less sensitivity for immunofluorescence than bench top analyzer flow cytometers.
• The MoFlo XDP can also physically sort up to four populations concurrently and it is able to sort them into a great variety of collection devices:
  
  • Microscope slides,
  • Microtubes,
  • 12 x 75 mm polypropelyne test tubes,
  • 14, 15 or 50 ml polypropelyne tubes,
  • Multiwell plates (24-, 96-, or 384-well plates, etc.; 60- or 72-well Terasaki, etc.),
  • A choice of a variety of custom devices that are only limited by the space constraints within the sort chamber area.
   

As mentioned previously, the MoFlo XDP has the same architecture and the 'original' MoFlo in the facility though the MoFlo XDP has 12, not 8, fluorescent detectors in the following configuration for immunophenotyping:

• The tunable argon laser is typically tuned to its 488-nanometer line (blue) and the argon laser is typically placed into the primary laser pathway which contains 7, not 6, optical detectors (2 optical detectors for laser light scatter and 5, not 4, for fluorescence detection),
• The tunable krypton laser is typically tuned to its multi-line violet mode (concurrent 407-, 413- and 415-nanometer lasing) and the krypton laser is typically placed into the secondary laser pathway that contains 3, not 2, fluorescence detectors and
• The tunable argon-krypton mixed-gas laser is typically tuned to its red 647-nanometer line and the mixed-gas laser is typically placed into the tertiary laser pathway that also contains 4, not 2, optical detectors. As mentioned previously, the MoFlo XDP is also equipped with a high-powered 592nm (orange) fiber laser, which shares the mixed-gas laser pathway.

Becton Dickinson FACSAria high-speed cell sorter flow cytometer [12 color, 4 laser system with optional 4-way sorting, aerosol containment, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking.

 

The core's FACSAria is an upgraded, 'Special Order' system that differs from the standard factory configuration that is equipped with 10 fluorescent detectors and 3 lasers. The core's FACSAria is equipped with 12, not 10, fluorescent detectors and 4, not 3, solid-state lasers as detailed in the following table. The core's FACSAria is equipped with an optional forward scatter photomultiplier tube (PMT) which is in consideration of investigators interested to sort particles <1 micron in size. The forward scatter PMT is in addition to the standard forward scatter diode, which exists in all the cytometers in the facility.

• The FACSAria, because it is a closed-cuvette sorter, has sensitivity equal to that observed in bench top analyzer flow cytometers, which is typically greater than the MoFlos.
• Like the MoFlos, the FACSAria can physically sort up to four populations concurrently and it is able to sort them into a great variety of collection devices:
  
  • Microscope slides,
  • Microtubes,
  • 12 x 75 mm test tubes,
  • 15 conical tubes,
  • Multiwell plates (24-, 96-, or 384-well plates, etc.; 60- or 72-well Terasaki, etc.),
   

The FACSAria is equipped with the following lasers in the detector configuration as described:

• Coherent Sapphire 20 milliwatt diode-pumped solid-state laser that emits blue light at 488 nanometers and which is connected to a detector bank configured in the octagonal configuration as the LSRII (see LSRII description). The FACSAria contains 8 PMTs (6 for fluorescence and 2 for laser light scatter) in the octagon.
• Coherent CUBE 40 milliwatt solid-state diode laser that emits red light at 638 nanometers and which is connected to a trigon that contains 2 fluorescent PMTs.
• Coherent CUBE 50 milliwatt solid-state diode laser that emits violet light at 405 nanometers and which is connected to a trigon that contains 3 fluorescent PMTs.
• Coherent Compass 40 milliwatt diode-pumped solid-state laser that emits yellow light at 561 nanometers and which is connected to a trigon that contains 2 fluorescent PMTs, all of which is detailed in the following table:

 

A 14-color, 5-laser FACSAria is available to investigators that is not physically located in the core facility though it is under the auspices and oversight of the core's administration and staff. The 5-laser FACSAria is equipped with the almost the identical lasers and forward scatter PMT that the 4-laser FACSAria has but the 5-laser FACSAria is also equipped with a green 532 nanometer laser. The lasers are and associated optics are:

• Coherent Sapphire 100 milliwatt diode-pumped solid-state laser that emits blue light at 488 nanometers and which is connected to an octagon that contains 6 PMTs (4 for fluorescence and 2 for laser light scatter).
• Coherent CUBE 40 milliwatt solid-state diode laser that emits red light at 638 nanometers and which is connected to a trigon that contains 3 fluorescent PMTs.
• Coherent CUBE 50 milliwatt solid-state diode laser that emits violet light at 405 nanometers and which is connected to a trigon that contains 2 fluorescent PMTs.
• Coherent Compass 25 milliwatt diode-pumped solid-state laser that emits yellow light at 561 nanometers and which is connected to a trigon that contains 2 fluorescent PMTs.
• Coherent Compass 150 milliwatt diode-pumped solid-state laser that emits green light at 532 nanometers and which is connected to a trigon that contains 3 fluorescent PMTs.

 

Differences in sorter model performance  

There are notable differences in performance between the disparate sorters in the facility that investigators that are sorting must be aware of. Known differences are detailed below. The take-home message should be that each model sorter in the facility has strengths and weaknesses that investigators should take into consideration when planning experiments that involve sorting.

FACSAria advantages:  

• The FACSAria is a closed-cuvette sorter that offers an advantage in fluorescence sensitivity over jet-in-air sorters such as the MoFlos. The fluorescence sensitivity of the FACSArias are equal to that observed on the bench top analyzer flow cytometers which also use a closed cuvette for fluorescence detection.

FACSAria disadvantages:  

• The FACSArias cannot sort as fast as the MoFlos.
• The sort yield (recovery) is lower on the FACSAria as compared to the MoFlos'. The FACSAria's yield is significantly reduced, due to aborted events, in its upper speeds (events/second) as compared to the MoFlos'.
• The FACSAria's available nozzle sizes (70 or 100μ, with a 130μ nozzle available as 'special order') are not nearly as broad a range as the MoFlos', which is 50-200μ, with company plans to reintroduce a 400μ nozzle, so the choice of sorting very large cells (particles) does not exist on FACSArias.
• Due to the open architecture of the MoFlos' design, the FACSArias are not as flexible in their configuration.
• Persons working with Zebrafish should know that the FACSAria can discern only 3 of the 5 major Zebrafish embryo laser light-scatter populations that are observed on the MoFlos.

As stated previously, the take-home message is that each model sorter in the facility has strengths and weaknesses that investigators must take into consideration when planning experiments that involve sorting.

iCys - Laser Scanning Cytometry (LSC)  

Introduction In the late 1990s, LSC has been developed that is capable of quantifying fluorescent and chromatic events in cells and tissues. LSC is a combination of microscopy and cytometry, all the principle (fluidic, optic and electronic) of flow cytometry basically can apply to laser scanning cytometry, except the fluidic is substituted by a moving stage of microscope. Since the data are acquired by microscopy lens, LSC provides not only quantitative ability of flow cytometry but also morphological images of microscopy.

Flow Cytometry Core Facility at Einstein recently acquired iCys equipped with a 405nm violet diode laser, a 488nm Argon laser and a 633nm Helium Neon laser from CompuCyte.

Below is the instrument and the optical configuration of our iCys.

 

 

6 sensors are installed in this iCys including 2 scatter sensors and 4 photomultipliers. The table below shows some fluorescent and chromatic dyes that can be detected by iCys.

Laser Excitation	Wavelength Band	Typical Fluorescent Dyes	Typical Fhromatic Dyes
405 nm	430 - 470 nm (blue)	DAPI, Hoechst33342, Pacific Blue, Quantum Dots	DAB, Permanent red, Noav Red
	515 - 545 nm (green)	Quantum Dots
	565 - 595 nm (orange)	Pacific Orange, Quantum Dots
	650 - 800 nm (long red)	Quantum Dots
488 nm	515 - 545 nm (green)	FITC, Alex488, Syto 16, YoPro	DAB, Permanent red, Nova Red, AEC
	565 - 595 nm (orange)	PE, Mitoshift
	650 - 800 nm (long red)	Pi, Pe-Cy5
633 nm	650 - 800 nm (long red)	Cy5, Alex647, Alex633, APC, DRAQ5	Hematoxinlin, BCIP

Advantages of using LSC LSC offers the following advantages over flow cytometry:

• Adherent cells could be examined in the culture plates (see the carrier section below) without trypsinization or scraping allowing the analysis with minimal disturbance of the cells;
• Time-resolved events in individual cells can be measured;
• Morphological images could be obtained at the same time of data acquisition;
• Subcellular localization of fluorochrome could be visualized;
• The sample could be re-analyzed or even re-stained;
• Retention of tissue architecture allows evaluation of cell populations;
• Hypocellular samples (fine needle aspirate) could be measured in multiparametric manner;
• Addition parameters (maximal pixel, peripheral integral, etc) are available to enhance the analysis. For example, besides G0/G1, S and G2/M phases typically resolved by propidium iodide in flow cytometry, two more populations, M-mitotic and post-mitotic could be separated by using the maximal pixel parameter with the same stain.

Carrier Cells could be analyzed in the following carrier:

• Microtiter plates. In general, microplates contain cellular or DNA samples. CompuCyte recommends the following types of microplates:
  96-well, plastic (such as Whatman cat# 7716-2380)
  96-well, glass, skirtless (such as Whatman #7716-2370)
  96-well, glass, skirted (such as Whatman #7716-2375)
  48-well, tissue culture-treated (such as BD Falcon #353078)
  24-well, tissue culture-treated (such as BD Falcon #353047)
  12-well, tissue culture-treated (such as BD Falcon #353043)
  6-well, tissue culture-treated (such as BD Falcon #353046)
  384-well, optical quality plastic (such as Whatman #7706-2103)
  384-well, optical quality plastic (such as Greiner #781096)
   
• Standard slide for both tissue and cellular samples.
• 35mm x 10mm culture dishes (such as Corning #430165).

Applications Below are some of the applications listed in the CompuCyte website or you may click into individual link to check out the details:

• Cell cycle 
• Apoptosis 
• DNA damage and repair 
• Translocation assays 
• Reporter assays 
• Live cell assays 
• Cell surface immunophenotyping 
• Tissue immunophenotyping 
• Tissue microarray analysis 

Using the LSC Individual who want to explore the instrument and to apply the technology in the research is encouraged to talk to the facility staffs.