EvErything counts

But nothing counts likE thE countEss® ii Fl AutomAtEd cEll countEr

2 Life Technologies™ | Countess® II FL Automated Cell Counter

The new Countess® II FL Automated Cell Counter is a benchtop cell assay platform equipped with state-of-the-art optics and image analysis software for rapid assessment of cells in suspension. With three-channel flexibility—brightfield and two optional fluorescence channels—researchers can count cells, monitor fluorescent protein expression, and measure cell viability.

•Flexibility—choose the fluorescence optical filters for your application

•Value—reusable counting slide reduces consumable costs

•Precision—autofocus designed to improve accuracy and reduce variability

•Simple—easy-to-use touch-screen interface

“I would describe the new Countess II® FL Automated Cell Counter as accurate, consistent, and effortless.”

ravi Pathak, Phd Baylor college of medicine

3Life Technologies™ | Countess® II FL Automated Cell Counter

Reusable or disposable slidesThe Countess® II FL Automated Cell Counter offers the option of using a reusable slide for counting cells, thereby significantly reducing long-term consumable costs. For those who prefer the convenience of disposable slides, the same slides that are used with the original Countess® Automated Cell Counter can also be used with the the Countess® II FL Automated Cell Counter (Figure 2).

Precise, accurate countsThe Countess® II FL Automated Cell Counter contains advanced autofocusing and counting algorithms to quickly and accurately identify and count cells within a population. Upon insertion of the slide, the Countess® II FL counter will automatically focus on the cells to avoid user variation associated with manual focusing mechanisms (Figure 3).

Clumpy cells? Not a problem with the Countess® II FL Automated Cell Counter. Whether using automated or manual cell counting methods, obtaining accurate cell counts in samples containing clumpy cells is difficult to achieve. The advanced counting algorithms of the Countess® II FL Automated Cell Counter can identify clear delineations of cell boundaries within clumps of cells, thereby giving precise, accurate cell counts even with clumpy samples (Figure 1).

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

NIH/3T3 HeLa HL-60

Countess® II FL

Hemocytometer

Figure 3. Precise, accurate cell counts with the Countess® II FL Automated Cell Counter. nih/3t3, hela, and hl-60 cells were mixed 1:1 in suspension with a 0.4% solution of trypan Blue stain (cat. no. 10282) and counted using the trypan blue exclusion method. 10 μl were added to a chamber slide (cat. no. c10228) and inserted into a countess® ii Fl Automated cell counter (blue bar) or hemocytometer and counted manually under a microscope (purple bar). Each bar is an average of 10 counts, with standard deviation as shown. the autofocusing and advanced counting algorithms of the countess® ii Fl Automated cell counter give a more precise, accurate cell count compared to manual counting.

Figure 1. Accurate cell counts—even with clumpy samples—on the Countess® II FL Automated Cell Counter. nih/3t3 cells were mixed 1:1 in suspension with a 0.4% solution of trypan Blue stain (cat. no. t10282), and a 10 μl sample was added to a chamber slide (cat. no. c10228) and inserted into a countess® ii Fl Automated cell counter. the segmentation and counting algorithms of the countess® ii Fl counter are able to resolve clumps of cells and score them as live or dead using the trypan blue exclusion method.

Figure 2. Reusable slide available for the Countess® II FL Automated Cell Counter.

4 Life Technologies™ | Countess® II FL Automated Cell Counter

Fluorescence counting that fits your experimentsThe Countess® II FL Automated Cell Counter uses the same fluorescent light cubes that power the EVOS® fluorescence imaging systems. EVOS® light cubes integrate LEDs and high-performance filters in a novel illumination system that delivers precise control, minimal maintenance, and exceptional reliability. With interchangeable light cubes, the Countess® II FL Automated Cell Counter was designed to evolve with your changing laboratory needs. As your experiments change, the fluorescence capabilities of the Countess® II FL counter can be upgraded with new EVOS® light cubes.

Full spectrum of fluorescence detectionWith over 15 EVOS® light cubes from which to choose, the Countess® II FL Automated Cell Counter is not limited to detection of GFP- and RFP-expressing cells (Table 1). Looking to measure the transfection efficiency of cells transduced with an RFP-expression construct with a blue nuclear counterstain? No problem. Simply add the RFP and DAPI light cubes, and you can effectively count the number of cells expressing the RFP fusion within the total population and accurately determine the transfection efficiency (Figures 4 and 5).

Figure 4. Two-color GFP and RFP fluorescent cell counting. 10 µl of hela cells transduced with celllight® histone 2B-rFP (c10595) and stained with nucBlue® live readyProbes® reagent were added to a chamber slide (c10228) and inserted into a countess® ii Fl Automated cell counter equipped with dAPi (AmEP4650) and rFP (AmEP4652) light cubes. the segmentation and counting algorithms of the new countess® ii Fl counter identifies and counts the cells based on their fluorescence properties. in this experiment, 86% of the hela cells within the population were transduced with the celllight® histone 2B-rFP construct.

Figure 5. Two-color GFP and DAPI fluorescent cell counting. 10 µl of hela cells transduced with celllight® mitochondria-gFP (c10600) and stained with nucBlue® live readyProbes® reagent were added to a chamber slide (c10228) and inserted into a countess® ii Fl Automated cell counter equipped with dAPi (AmEP4650) and gFP (AmEP4651) light cubes. the segmentation and counting algorithms of the new countess® ii Fl counter identifies and counts the cells based on their fluorescence properties. in this experiment, 72% of the hela cells within the population were transduced with the celllight® mitochondria-gFP construct.

5Life Technologies™ | Countess® II FL Automated Cell Counter

EVOS® light cubes for the Countess® II FL Automated Cell CounterAt the heart of the Countess® fluorescence technology lie the proprietary EVOS® LED light cubes. Each cube contains an LED, illuminating optics, and filters. Light cubes are user interchangeable, auto-configured by the Countess® system with plug-and-play capability. The wide variety of light cubes available provides flexibility for multiple-fluorescence research applications, like cell viability analysis (Figure 6).

Figure 6. Cell viability. heat-treated hl-60 cells were combined with non-treated cells at approximately a 15:85 ratio. to assess cell viability of the population, cells were stained with a combination of nucBlue® live and nucgreen® dead 488 stains. 10μl of cells were added to a chamber slide and inserted into a countess® ii Fl Automated cell counter equipped with dAPi (AmEP4650) and gFP (AmEP4651) light cubes. As expected, 14% of the hl-60 cell population were dead as indicated by positive nucgreen® dead 488 (green) staining.

Light cube Excitation (nm) Emission (nm) Common compatible dyes/fluorescent proteins

dAPi 357/44 447/60 DAPI, Hoechst, BFP

tagBFP 390/18 447/60 TagBFP

cFP 445/45 510/42 ECFP, Lucifer Yellow

gFP 470/22 510/42 GFP, Alexa Fluor® 488, SYBR® Green, FITC

yFP 500/24 524/27 EYFP, acridine orange (+DNA)

rFP 531/40 593/40 RFP, Alexa Fluor® 546, Alexa Fluor® 555, Cy®3, DsRed, Rhodamine Red, dTomato

texas red® 585/29 624/40 Texas Red®, Alexa Fluor® 568, Alexa Fluor® 594, MitoTracker® Red, mCherry

cy®5 628/40 692/40 Cy®5, Alexa Fluor® 647, Alexa Fluor® 660, DRAQ5®

cy®5.5 655/46 794/16 Cy®5.5

For a complete list of available common and specialty EVOS® light cubes, go to lifetechnologies.com/evoslightcubes

Table 1. Common fluorescent light cubes available for the Countess® II FL Automated Cell Counter

6 Life Technologies™ | Countess® II FL Automated Cell Counter

Cell type Animal Organ

A431 human skin

Adipocytes human Adipose-derived stem cells

Aortic smooth muscle human smooth muscle

cho-m1Wt2 chinese hamster ovary

chsE chinook salmon Embryo

colo-205 human colon

cos-7 African monkey kidney

hEk-293 human kidney

hela human cervix

hepg2 human liver

hl-60 human Blood

J774A.1 mouse Blood

Jurkat human Blood

k562 human Bone marrow

mcF7 human Breast

mrc-5 human lung

nih/3t3 mouse Embryo

Pc-12 rat Adrenal gland

Pulmonary artery endothelial cells human Blood vessel

Pulmonary artery smooth muscle human smooth muscle

sF-21 insect ovary

u266 human Blood

u-2 os human Bone

umbilical vein endothelial cells human Blood vessel

Table 2. Cell lines validated on the Countess® II FL Automated Cell Counter

7Life Technologies™ | Countess® II FL Automated Cell Counter

technical specifications

Processing time: <10 seconds

sample concentration range: 1 × 104 to 1 × 107 cells/ml

Particle/cell diameter range: 10–60 μm

required sample volume: 10 μl

Firmware: countess® Automated cell counting Platform

usB drive: software

opticsoptics: 3 channels (brightfield and 2 slots for Evos® lEd light cubes)

camera: 5 megapixels, 2.5x optical magnification

Physical characteristics

instrument type: Benchtop cell counter and suspension cell–based assay platform

instrument dimensions:9" (W) × 5½" (d) × 9" (h)228.6 mm (W) X 139.7 mm (d) x 228.6 mm (h)

Weight: 8 lb

operating power: 100–240 vAc, 0.58 A max

Frequency: 50/60 hz

Electrical input: 12 vdc, 2 A

installation site:indoor use only, class A environments (i.e., nonresidential or light industrial); Pollution degree 2.

operating temperature: 10–40˚c

operating humidity: <80% (noncondensing)

reusable analysis slide

material: glass

dimensions:slide: 32 mm (W) × 76 mm (d) × 3.9 mm (h) coverslip: 20 mm (W) x 26 mm (l) x 0.5 mm (h)

chamber volume: 10 μl

disposable analysis slide

material: Poly(methyl methacrylate) (PmmA)

dimensions: slide: 25 mm (W) × 75 mm (d) × 1.7 mm (h)

chamber volume: 10 μl

Table 3. Countess® II FL Automated Cell Counter technical specifications

Find out more at lifetechnologies.com/countessII

For Research Use Only. Not for use in diagnostic procedures. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Cy is a registered trademark of GE Healthcare. DRAQ5 is a registered trademark of Biostatus Limited. CO012822 1214

Need a simple, brightfield cell counter? Check out the Countess® II Automated Cell Counter.

Feature Countess® II Countess® II FL

Autofocus ✔ ✔

touch-screen ✔ ✔

Brightfield counting ✔ ✔

trypan blue counting ✔ ✔

disposable slide ✔ ✔

reusable slide ✔

Fluorescence compatible/upgradable ✔

uses Evos® light cubes ✔

Find out more at lifetechnologies.com/countessII

Ordering information

Product Quantity Cat. No.

countess® ii Fl Automated cell counter 1 AmQAF1000

countess® ii Automated cell counter 1 AmQAX1000

countess® cell counting chamber slides 50 c10228

countess® ii Fl reusable slide 1 A25750

countess® ii Fl reusable slide holder 1 AmEP4746

Evos® light cube, dAPi 1 AmEP4650

Evos® light cube, tagBFP 1 AmEP4668

Evos® light cube, yFP 1 AmEP4654

Evos® light cube, gFP 1 AmEP4651

Evos® light cube, rFP 1 AmEP4652

Evos® light cube, texas red® 1 AmEP4655

Evos® light cube, cy®5 1 AmEP4656

Evos® light cube, cy®5.5 1 AmEP4673