STEMvision™

Automated and standardized colony counting for the hematopoietic colony-forming unit (CFU) assay

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Automated and standardized colony counting for the hematopoietic colony-forming unit (CFU) assay

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Overview

STEMvision™ consists of an instrument and software designed specifically for imaging and counting colonies in hematopoietic colony-forming unit (CFU) assays using MethoCult™ media and meniscus-free SmartDish™ cultureware. STEMvision™ software, known as Analysis Packages, replaces the need to manually count colonies using a microscope.

For human cultures, three Analysis Packages have been designed for scoring and counting hematopoietic colonies produced by erythroid, myeloid and multi-potential progenitor cells in 14-day assays of CB, BM and MPB cultured in MethoCult™ Optimum media. A fourth Analysis Package is also available for counting the total number of colonies in 7-day assays of CB cultured in MethoCult™ Express medium.

For mouse BM cultures, three Analysis Packages have been designed to count total numbers of hematopoietic colonies produced by all combined myeloid and erythroid progenitors in M3434, myeloid progenitors only in M3534 and erythroid progenitors only in M3436.

Selection of at least one Analysis Package is required for instrument purchase. Additional Analysis Packages are sold separately for labs that currently own a STEMvision™ instrument.

Select your preferred Analysis Package and Request Pricing for further information. Several purchase and leasing options are available.

For more information about Instrument Services including additional service packages and installation software please see our instrumentation overview.
Application:
Colony Assay
Brand:
STEMvision
Area of Interest:
Stem Cell Biology; Cord Blood Banking

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This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Representative STEMvision™ Images Showing Colonies Derived from CB Progenitors after 7 Days of Culture in MethoCult™ Express, and from CB, BM and MPB after 14 Days of Culture in MethoCult™ Optimum

Figure 1. Representative STEMvision™ Images Showing Colonies Derived from CB Progenitors after 7 Days of Culture in MethoCult™ Express, and from CB, BM and MPB after 14 Days of Culture in MethoCult™ Optimum

These images have been analyzed by STEMvision™ Human (A) 7-Day and (B-D) 14-Day Analysis Packages. Green circles identify individual colonies in a 7-day CB CFU assay that scores total CFUs only (A). Orange and red circles identify erythroid colonies, yellow circles identify myeloid colonies and blue circles identify mixed colonies in 14-day CB (B), BM (C) and MPB (D) CFU assays. Erythroid and mixed colonies that contain hemoglobinized cells are shown in true red color.

STEMvision™ Automated Counting of Total, Erythroid (BFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day CB CFU Assays

Figure 2. STEMvision™ Automated Counting of Total, Erythroid (BFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day CB CFU Assays

Cryopreserved CB cells were thawed, plated in MethoCult™ Optimum and cultured for 14 days. The resulting colonies were then counted both manually using an inverted microscope, and automatically using STEMvision™ with the Human CB 14-Day CFU Analysis Package (Catalog #22005). Gray dashed lines represent a perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The mathematical equations and correlation coefficients (R2) that describe each data set (n=130 CFU assays) are as follows: A: y=1.02x + 1.39, R2=0.96 for Total Colonies, B: y=1.05x + 1.53, R2=0.89 for BFU-E, C: y=0.99x + 0.13, R2=0.94 for CFU-G/M/GM.

STEMvision™ Automated Counting of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day CB CFU Assays

Figure 3. STEMvision™ Automated Counting of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day CB CFU Assays

Thirty individual 14-day CB CFU assays were counted by three to seven people. The numbers of mixed colonies (CFU-GEMM) colonies counted manually in each well are shown by the black open circles (n=80 total assay scores). Manual CFU-GEMM counts in most cultures varied significantly between individual people. STEMvision™ counts of the same cultures (red circles) provided a CFU-GEMM count that was typically within the range of manual counts.

STEMvision™ Automated Scoring of Total, Erythroid (BFU-E + CFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day BM CFU Assays

Figure 4. STEMvision™ Automated Scoring of Total, Erythroid (BFU-E + CFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day BM CFU Assays

Cryopreserved BM cells were thawed, plated in MethoCult™ Optimum, cultured for 14 days, and the resulting colonies then scored both manually using an inverted microscope and automatically using STEMvision™ with the Human BM 14-Day CFU Analysis Package (Catalog #22006). The BM Analysis Package can identify and count erythroid colonies produced by CFU-E and BFU-E separately, but these are combined in panel B. Gray dashed lines represent a perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The mathematical equations and correlation coefficients (R2) that describe each data set (n=120 CFU assays) are as follows: A: y=0.88x + 8.79, R2=0.95 for Total Colonies, B: y=0.83x + 6.71, R2=0.89 for CFU-E + BFU-E, C: y=0.92x + 2.55, R2=0.94 for CFU-G/M/GM.

STEMvision™ Automated Counting of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day BM CFU Assays

Figure 5. STEMvision™ Automated Counting of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day BM CFU Assays

Thirty individual 14-day BM CFU assays were counted by three to seven people. The numbers of mixed (CFU-GEMM) colonies counted manually in each well is shown by the black open circles (n=82 total assay scores). Manual CFU-GEMM counts in most cultures varied significantly between individual people. STEMvision™ counts of the same cultures (red circles) provided a CFU-GEMM count that was typically within the range of manual counts.

STEMvision™ Automated Counting of Total, Erythroid (BFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day MPB CFU Assays

Figure 6. STEMvision™ Automated Counting of Total, Erythroid (BFU-E) and Myeloid (CFU-G/M/GM) Colonies Is Highly Correlated to Manual Counts of 14-Day MPB CFU Assays

Cryopreserved MPB cells were thawed, plated in MethoCult™ Optimum, cultured for 14 days, and the resulting colonies then scored both manually using an inverted microscope and automatically using STEMvision™ with the Human MPB 14-Day CFU Analysis Package (Catalog #22007). Gray dashed lines represent a perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The mathematical equations and correlation coefficients (R2) that describe each data set (n=143 CFU assays) are as follows: A: y=0.97x + 2.44, R2=0.97 for Total Colonies, B: y=0.96x + 3.74, R2=0.91 for BFU-E, C: y=0.96x + 0.90, R2=0.95 for CFU-G/M/GM.

STEMvision™ Automated Scoring of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day MPB CFU Assays

Figure 7. STEMvision™ Automated Scoring of Mixed Colonies Falls Within the Range of Manual Counts of 14-Day MPB CFU Assays

Thirty individual 14-day MPB CFU assays were counted by three to seven people. The numbers of mixed (CFU-GEMM) colonies counted manually in each well is shown by the black open circles (n=82 total assay scores). Manual CFU-GEMM counts in most cultures varied significantly between individual people. STEMvision™ counts of the same cultures (red circles) provided a CFU-GEMM count that was typically within the range of manual counts.

Representative STEMvision™ Images Showing Colonies Derived from Mouse BM Progenitors After 12 Days of Culture in MethoCult™ GF M3434, MethoCult™ GF M3534 or MethoCult™ SF M3436 Media

Figure 8. Representative STEMvision™ Images Showing Colonies Derived from Mouse BM Progenitors After 12 Days of Culture in MethoCult™ GF M3434, MethoCult™ GF M3534 or MethoCult™ SF M3436 Media

Images of mouse BM cells cultured in (A) MethoCult™ GF M3434, (B) MethoCult™ GF M3534 and (C) MethoCult™ SF M3436 were acquired using STEMvision™. The corresponding STEMvision™ Mouse Analysis Package (Table 1) was used to analyze each image. Red circles identify the smallest colonies - size class 1, yellow circles - size class 2, blue circles - size class 3 and orange circles identify the largest colonies - size class 4.

STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Total (Myeloid Plus Erythroid) Colonies in Mouse BM CFU Assays

Figure 9. STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Total (Myeloid Plus Erythroid) Colonies in Mouse BM CFU Assays

BM cells were plated in MethoCult™ GF M3434 (Catalog #03434/03444). Colonies were counted on days (A) 7, (B) 10 and (C) 12 both manually using an inverted microscope, and automatically using STEMvision™ equipped with the Mouse Total CFU Analysis Package (Catalog #22008). We recommend counting CFU assays of mouse progenitor cells plated in M3434 between 10 and 12 days. Gray dashed lines represent a theoretical perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The slope, 95% confidence intervals, correlation coefficients (R2) and sample size for each data set are provided in Table 1.

STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Myeloid Colonies in Mouse BM CFU Assays

Figure 10. STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Myeloid Colonies in Mouse BM CFU Assays

BM cells were plated in MethoCult™ GF M3534 (Catalog #03534). Colonies were counted on days (A) 7, (B) 10 and (C) 12 both manually using an inverted microscope, and automatically using STEMvision™ equipped with the Mouse Myeloid CFU Analysis Package (Catalog #22009). We recommend counting CFU assays of mouse myeloid progenitor cells plated in M3534 between 10 and 12 days. Gray dashed lines represent a theoretical perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The slope, 95% confidence intervals, correlation coefficients (R2) and sample size for each data set are provided in Table 1.

STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Erythroid Colonies in Mouse BM CFU Assays

Figure 11. STEMvision™ Automated Counting is Highly Correlated to Manual Counting of Erythroid Colonies in Mouse BM CFU Assays

BM cells were plated in MethoCult™ SF M3436 (Catalog #03436). Colonies were counted on days (A) 7, (B) 10 and (C) 12 - 14 both manually using an inverted microscope, and automatically using STEMvision™ equipped with the Mouse Erythroid CFU Analysis Package (Catalog #22011). We recommend counting CFU assays of mouse erythroid progenitor cells plated in M3436 between of 10 to 14 days. Gray dashed lines represent a theoretical perfect linear correlation between manual and automated counts. Red solid lines represent the actual linear correlation between manual and automated counts. The slope, 95% confidence intervals, correlation coefficients (R2) and sample size for each data set are provided in Table 1.

Table 1. Correlation Between Automated STEMvision™ and Manual Colony Counting

Table 1. Correlation Between Automated STEMvision™ and Manual Colony Counting

*CI: Confidence Internal
**Mouse CFU assays of erythroid progenitor cells plated in M3436 should be counted between 10 to 14 days.

Publications

(1)
The Journal of experimental medicine 2014 AUG

RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development.

Verma R et al.

Abstract

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated textgreater20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.
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