CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 1	Human	Low serum	KCO-HPP1-1H	1 Kit
HPP-SP 2	Human	Low serum	KCO-HPP2-1H	1 Kit
CFC-GEMM 1	Human	Low serum	KCO-GEMM1-1H	1 Kit
CFC-GEMM 2	Human	Low serum	KCO-GEMM2-1H	1 Kit
CFC-GEMM 3	Human	Low serum	KCO-GEMM3-1H	1 Kit
CFC-GEM 1	Human	Low serum	KCO-GEM1-1H	1 Kit
CFC-GEM 2	Human	Low serum	KCO-GEM2-1H	1 Kit
CFC-GEM 3	Human	Low serum	KCO-GEM3-1H	1 Kit
Progenitor Cell Populations
BFU-E 1	Human	Low serum	KCO-BFU1-1H	1 Kit
GM-CFC 1	Human	Low serum	KCO-GM1-1H	1 Kit
GM-CFC 2	Human	Low serum	KCO-GM2-1H	1 Kit
Mk-CFC 1	Human	Low serum	KCO-Mk1-1H	1 Kit
T-CFC	Human	Low serum	KCO-T-1H	1 Kit
No Growth Factors Added
Any	Human	Low serum	KCO-0-1H	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 1	Mouse	Low serum	KCO-HPP1-1M	1 Kit
HPP-SP 2	Mouse	Low serum	KCO-HPP2-1M	1 Kit
CFC-GEMM 1	Mouse	Low serum	KCO-GEMM1-1M	1 Kit
CFC-GEMM 2	Mouse	Low serum	KCO-GEMM2-1M	1 Kit
CFC-GEM 1	Mouse	Low serum	KCO-GEM1-1M	1 Kit
CFC-GEM 3	Mouse	Low serum	KCO-GEM3-1M	1 Kit
Progenitor Cell Populations
BFU-E 1	Mouse	Low serum	KCO-BFU1-1M	1 Kit
GM-CFC 1	Mouse	Low serum	KCO-GM1-1M	1 Kit
Mk-CFC 1	Mouse	Low serum	KCO-Mk1-1M	1 Kit
T-CFC	Mouse	Low serum	KCO-T-1M	1 Kit
No Growth Factors Added
Any	Mouse	Low serum	KCO-0-1M	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Primate	Low serum	KCO-HPP2-1Pr	1 Kit
CFC-GEMM 1	Primate	Low serum	KCO-GEMM1-1Pr	1 Kit
Progenitor Cell Populations
BFU-E 1	Primate	Low serum	KCO-BFU1-1Pr	1 Kit
GM-CFC 1	Primate	Low serum	KCO-GM1-1Pr	1 Kit
Mk-CFC 1	Primate	Low serum	KCO-Mk1-1Pr	1 Kit
T-CFC	Primate	Low serum	KCO-T-1Pr	1 Kit
No Growth Factors Added
Any	Primate	Low serum	KCO-0-1Pr	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Rat	Low serum	KCO-HPP2-1R	1 Kit
CFC-GEMM 1	Rat	Low serum	KCO-GEMM1-1R	1 Kit
Progenitor Cell Populations
BFU-E 1	Rat	Low serum	KCO-BFU1-1R	1 Kit
GM-CFC 1	Rat	Low serum	KCO-GM1-1R	1 Kit
Mk-CFC 1	Rat	Low serum	KCO-Mk1-1R	1 Kit
T-CFC	Rat	Low serum	KCO-T-1R	1 Kit
No Growth Factors Added
Any	Rat	Low serum	KCO-0-1R	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Dog	Low serum	KCO-HPP2-1C	1 Kit
CFC-GEMM 1	Dog	Low serum	KCO-GEMM1-1C	1 Kit
Progenitor Cell Populations
BFU-E 1	Dog	Low serum	KCO-BFU1-1C	1 Kit
GM-CFC 1	Dog	Low serum	KCO-GM1-1C	1 Kit
Mk-CFC 1	Dog	Low serum	KCO-MK1-1C	1 Kit
No Growth Factors Added
Any	Dog	Low serum	KCO-0-1C	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Horse	Low serum	KCO-HPP2-1E	1 Kit
CFC-GEMM 1	Horse	Low serum	KCO-GEMM1-1E	1 Kit
Progenitor Cell Populations
BFU-E 1	Horse	Low serum	KCO-BFU1-1E	1 Kit
GM-CFC 1	Horse	Low serum	KCO-GM1-1E	1 Kit
Mk-CFC 1	Horse	Low serum	KCO-Mk1-1E	1 Kit
No Growth Factors Added
Any	Horse	Low serum	KCO-0-1E	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Pig	Low serum	KCO-HPP2-Po	1 Kit
CFC-GEMM 1	Pig	Low serum	KCO-GEMM1-Po	1 Kit
Progenitor Cell Populations
BFU-E 1	Pig	Low serum	KCO-BFU1-Po	1 Kit
GM-CFC 1	Pig	Low serum	KCO-GM1-Po	1 Kit
Mk-CFC 1	Pig	Low serum	KCO-Mk1-Po	1 Kit
T-CFC	Pig	Low serum	KCO-T-Po
No Growth Factors Added
Any	Pig	Low serum	KCO-0-Po	1 Kit

CAMEO™-96

A Standardized Proliferation and Differentiation Methylcellulose CFU Assay in a 96-Well Plate Format

Cell Population	Species	Formulation	Catalog Number	Quantity
Stem Cell Populations
HPP-SP 2	Sheep	Low serum	KCO-HPP2-1O	1 Kit
CFC-GEMM 1	Sheep	Low serum	KCO-GEMM1-1O	1 Kit
Progenitor Cell Populations
BFU-E 1	Sheep	Low serum	KCO-BFU1-1O	1 Kit
GM-CFC 1	Sheep	Low serum	KCO-GM1-1O	1 Kit
Mk-CFC 1	Sheep	Low serum	KCO-Mk1-1O	1 Kit
No Growth Factors Added
Any	Sheep	Low serum	KCO-0-1O	1 Kit

 

• Standardized the methylcellulose CFU assay.
• Available to standardized human CFU assays, equivalent to Methocult assays, for cellular therapy applications.
• All applications involving lympho-hematopoietic cells with a manual or image analysis readout.
• Leukemic cell proliferation and differentiation.
• Detect clonal ability of cells.
• Evaluate the differentiation status or potential of different lympho-hematopoietic cell populations.
• Correlate in vivo with in vitro differentiation.
• Differentiation effects of growth factors or cytokines.
• Evaluate ex vivo differential expansion.
• Evaluate the effects of drugs, xenobiotics or other agents on the differentiation/maturation process.  

• CAMEO™-96 is the only complete assay in which both proliferation and differentiation can be determined simultaneously, using different readouts.
• Allows standardization of the CFU/CFC assay using standards and controls.
• Allows conversion of colony counts into standardized and quantitative ATP concentrations.
• Complete flexibility; evaluate growth factors or cytokines.
• Available for 19 different cell populations and 8 species.
• Incorporates powerful ATP bioluminescence readout technology to measure cell proliferation.

 

All ATP bioluminescence assays kits from Preferred Cell Systems™ include an ATP standard(s) and controls.  Performing the ATP standard curve and controls is highly recommended. The controls are used to calibrate the luminescence plate reader and the ATP standard is used to standardize the assay. The values obtained from the standard curve and controls (see diagram below) are then compared to the measurement assurance parameters that are provided in the instruction manual. If the values obtained are within the ranges provided by the measurement assurance parameters, you, the user, have successfully performed the proficiency testing to ensure that you have not only performed the assay correctly, but that the results you obtain can be trusted.

No additional proficiency testing is required if the calibration and standardization procedure is performed. The values you obtain from each calibration and standardization can be logged and used for certification that the assay has been performed correctly and that the results are trustworthy.


 

• Human
• Non-human primate (Macaca fascicularis (Cynomologus) and Macaca mulatta (Rhesus))
• Horse
• Pig
• Sheep
• Dog
• Rat
• Mouse

The following cell populations are available as CAMEO™-96 Complete Assay Kits.  Those populations that have multiple growth factors will produce different colony types in the same culture. Please note that the number of colonies counted and their proliferation in cultures containing multiple growth factors will be different to those with single growth factors, which only stimulate cells in a specific cell compartment.
 

Cell Type	Cell Population Detected (1)	MethoCult™ Equivalent(2)	Growth Factor Cocktail	Suggested Number of Samples(3)
Any	None	NE	None	24, 16 or 12
Stem cell	HPP-SP 1	NE	3,6,SCF,Flt3-L	24, 16 or 12
Stem cell	HPP-SP 2	NE	EPO,GM,2,3,6,7,SCF,TPO, Flt3-L	24, 16 or 12
Stem cell	CFC-GEMM 1	"Express"	EPO,GM,3,6,SCF,TPO, Flt3-L	24, 16 or 12
Stem cell	CFC-GEMM 2	NE	EPO,GM,3,6,SCF	24, 16 or 12
Stem cell	CFC-GEMM 3(4)	H4435 "Enriched"	EPO,GM,G,3,6,SCF + TPO	24, 16 or 12
Stem cell	CFC-GEM 1	NE	EPO,GM3,6,SCF	24, 16 or 12
Stem cell	CFC-GEM 2	H4434 "Classic"	EPO,GM,G,3,SCF	24, 16 or 12
Stem cell	CFC-GEM 3(4)	H4034 "Optimum"	EPO,GM,G,3,SCF	24, 16 or 12
Progenitor	BFU-E 1	NE	EPO,3,SCF	24, 16 or 12
Progenitor	GM-CFC 1	H4534 "Classic"	GM,3,SCF	24, 16 or 12
Progenitor	GM-CFC 2	H4035 "Optimum"	GM,G,3,SCF	24, 16 or 12
Progenitor	Mk-CFC 1	NE	TPO,3,SCF	24, 16 or 12
Progenitor	T-CFC	NE	2 alone	24, 16 or 12
Progenitor	B-CFC(5)	NE	7 alone	24, 16 or 12

NE = No equivalent product available.
(1) = Accepted nomenclature of cell populations detected by the colony-forming cell assay.
(2) = Human MethoCult equivalent assay for cellular therapy applications.
(3) = The number of samples is based on the number of replicate wells/sample. If 4 replicate wells/sample are used, 24 samples/plate can be detected. Using 6 replicate wells/sample, 16 samples can be detected and for 8 replicates wells/samples prepared in columns, 12 samples can be measured.
(4) = Only available for human.
(5) = Available for primate, dog, rat and mouse only.

Preferred Cell Systems™ provides 4 different colony-forming unit assays in 3 different formats. 

CAMEO™-96 is one of the 4 CFU assays available in a 35mm Petri dish format. Below, it is compared to the other 3 CFU assays available from Preferred Cell Systems™.

Characteristics	AllColonies™: Traditional/ Micro/Multi	ColonyGro™	CAMEO™-4	CAMEO™-96
Uses:	Basic and stem cell research. Veterinary research. Limited cellular therapy.	Basic and stem cell research. Veterinary research. Limited cellular therapy.	Basic and stem cell research. Veterinary research. Limited cellular therapy.	Basic and stem cell research. Veterinary research. Limited cellular therapy. Limited hematotoxicity testing
Assay availability:	100mL/50mL/1kit	100mL	50mL	1 kit
Number of samples:	44/50/Depends on number of replicates required(1)	44	50	Depends on number of replicates required(1)
Type of assay:	Methylcellulose  - Clonal	Methylcellulose - Clonal	Methylcellulose - Clonal	Methylcellulose - Clonal
Assay format:	35mm Petri dish/ 4-well Petri dish/ 96-well plate	35mm Petri dish	4-well Petri dish	96-well plate
Cell functionality detected:	Differentiation or cell differentiation AND proliferation	Differentiation	Differentiation	Cell differentiation AND proliferation
Readout:	Microscopic or microscopic AND plate reader	Microscopic	Microscopic	Microscopic AND plate reader
Readout endpoint:	Colony counts or colony counts WITH ATP bioluminescence	Colony counts	Colony counts	Colony counts WITH ATP bioluminescence
Subjectivity:	Subjective/subjective/ non-subjective	Subjective	Subjective	Subjective AND non-subjective
Cell types detected:	Primitive lympho- hematopoietic stem cells	Stem, progenitor and/or precursor cells	Stem, progenitor and/or precursor cells	Stem and/or progenitor cells
Species:	Human. Primate. Dog. Rat. Mouse	Human. primate. Horse. Pig. Sheep. Dog. rat. Mouse	Human. primate. Horse. Pig. Sheep. Dog. rat. Mouse	Human. primate. Horse. Pig. Sheep. Dog. rat. Mouse
MethoCult™ equivalent assays available:	No	Yes	Yes	Yes
Total culture volume required:	2.5mL/0.6mL/ Depends on replicate number (2)	2.5mL	0.6mL	Depends on replicate number (2)
Number of replicates/culture:	2/4/As many as required	2	4	As many as required
Volume/culture:	1mL/0.1mL/0.1mL	1mL	0.1mL	0.1mL
Cell culture time(3):	6-10 days	2-12 days	2-9 days	4-9 days
Instrument calibration:	Only for AllColonies™ Multi(4)	No	No	Yes(4)
Standardized assay (5):	Only for AllColonies™ Multi	No	No	Yes
Proficiency testing incorporated into assay(6):	Only for AllColonies™ Multi	No	No	Yes
Measurement assurance(7):	Only for AllColonies™ Multi	No	No	Yes
Validation(8):	Only for AllColonies™ Multi	No	No	Yes

(1) = The number of samples tested depends on the number of replicates/sample performed. For example, if 6 replicates/sample are performed, then 16 samples can be analyzed on a single 96-well plate. These samples, however, do not have to performed on the same day.
(2) = The total culture volume for AllColonies™ Multi and CAMEO™-96 depends on the number of replicates/sample performed. If 4 replicates are required, 0.6mL total volume would be prepared, while for 6 replicates, 0.8mL would be prepared.
(3) = Culture time depends on the the state of fractionation or purification of the source cells, cell population tested and species.
(4) = Instrument calibration is performed using the ATP controls included with the assay kit.
(5) = Standardization implies performing a standard curve. ATP standards are included. ColonyGro™ and CAMEO™-4 or any other methylcellulose colony-forming assay from competitors do not include standards or controls and can not be standardized. "Standardized media" does not make an assay "standardized". 
6 = Since AllColonies™ Multi and CAMEO™-96 include standards and controls that allow the results to be compared to measurement assurance parameters every time an assay is performed, proficiency testing is also performed automatically at the same time as the instrument is calibrated and the assay standardized.
7 = Measurement assurance parameters are included in the Instruction Manual.
8 = Validation parameters are included in the Instruction Manual. Since standards and controls are included with these assays, validation can be performed in-house. ColonyGro™ and CAMEO™-4 as well as all other methylcellulose colony-forming assays from competitors, do not include standards and controls and, therefore, cannot be standardized or validated.

The graph below shows how the methylcellulose CFU assay is standardized. (See Common Questions on CAMEO™-96, below). The steps to standardize any CFU are as follows:

• Set up a CAMEO™-96 assay with difference cell concentrations.
• After cell culture, manually count the number of colonies for each cell dilution for all replicate wells.
• Calibrate and standardize the ATP bioluminescence step of the assay with the ATP standard and calibrators included with the assay kit.
• Ensure that the slope of the ATP standard curve and the calibration controls are within the range specified in the Instruction Manual.
• Process all wells of the assay by adding 0.1mL of the ATP-ER reagent, mix, and leave to 10 min in the dark prior to measuring the bioluminescence in a plate luminometer.
• Plot the number of colonies counted against the reported ATP concentration.
• Convert the number of colonies to standardized ATP concentrations as shown in the graph below.
• For unknown samples, interpolate the number of colonies counted into standardized ATP concentrations.

By performing this procedure, you have not only standardized the CFU assay, but have also measured the proliferation of cells present in differentiating colonies of cells.

• CAMEO™-96 Reagent
• ATP standard
• ATP high and low controls
• ATP Enumeration Reagent
• Sterile, 96-well plates
• Non-sterile-96-well plates
• Sterile, adhesive foil covers

What is the difference between CAMEO-™4 and CAMEO™-96?
There are many differences between these 2 assays. CAMEO™-4 is a traditional miniaturized methylcellulose colony-forming assay. CAMEO™-96 is also a methylcellulose colony forming assay, but is performed in a clear bottom, 96-well plate in which colonies can be counted and cell proliferation within the colonies measured using the most sensitive and accurate measurement of cell proliferation available, namely ATP bioluminescence. CAMEO™-96 also distinguishes itself from all other methylcellulose assays it that the colony-forming cell (CFC) assay is fully standardized. Standardization of the CFC assay is otherwise impossible with traditional methylcellulose assay.
 
How is the CAMEO™-96 methylcellulose CFC assay standardized?
After cell culture, the colonies are counted under an inverted microscope. Prior to measuring cell proliferation, this second step of the assay is calibrated and standardized against an ATP standard. The cultures are then processed and the amount of cell proliferation is determined in a luminescence plate reader. The amount of proliferation correlated directly with the concentration of intracellular ATP produced by the cells. When the total number of colonies is plotted against the ATP concentration, the number of colonies can be directly interpolated into standardized ATP concentrations. Since the ATP step of the assay is standardized, the colony counting step is also standardized.
 
How many replicate wells are needed for a CAMEO™-96 assay?
Preferred Cell Systems™ recommends a minimum number of 4 replicate wells per data point. Six replicate wells are better from a statistical viewpoint. However, the investigator is flexible to use any number of replicate wells they choose.
 
What species and cell populations are available with CAMEO™-96?
CAMEO™-96 assay kits are available for human, primate, dog rat and mouse cells. The assay kits are available for up to 11 different populations from each species.
 
How long does CAMEO™-96 take to perform?
This depends on the species. However, in general, CAMEO™-96 for human cells is a 7 day assay, while that for all other species is a 5 day assay. This is much faster than for any traditional CFC assay.
 
How long does the ATP bioluminescence step take?
The calibration and standardization of the assay will take between 20-30min to prepare and read the results. A full plate of samples will take about 15-20min to process, followed by a 10min incubation in the dark and less than 5 min to measure.
 
Is there a non-methylcellulose version of CAMEO™-96?
Yes. The non-methylcellulose version of CAMEO™-96 is called HALO® and incorporate Suspension Expansion Culture™ (SEC™) Technology.
 

Please note: Certificate of Analysis (CoAs) sheets will be provided with the assay kit
Download the CAMEO™-96 Technical Manual
Download the ATP Optimization Kit Protocol for First-Time Users
Download Luminometer Setup and RLU to ATP Conversion
Download methylcellulose SDS sheet
Download ATP standards and controls SDS sheet
Download ATP Enumeration Reagents (ATP-ER) SDS sheet
Download SEC™ Media SDS sheet