connectionconnectionW

inter 2004

If your genomics research involves high-throughput screening methods, purchas-

ing entire plates of clones or clone sets may be the fastest and most cost-effective way to achieve results.

ATCC has full-length, putative full-length, and EST plates from the IMAGE Consortium as well as other sets and collections. The tables on page 3 describe plates that are currently available. New plates arrive reg-ularly and MGC plates are updated peri-odically as new sequences are submitted. Detailed information on IMAGE plates can be found at http://image.llnl.gov/.

Clones from the Mammalian Gene Col-lection (MGC) have fully sequenced open reading frames (ORFs) and are guaranteed

to be correct. These high-quality clones are distributed in 96-well plates sorted by vector type.

Putative MGC plates are the candi-date plates from which the MGC full-length clones are derived. About 60% of these clones are found to be full length. Each plate contains clones from multiple libraries but from one species and with one resistance marker.

EST plates contain the least charac-terized clones, thousands of which are isolated at random from a given cDNA library. Only the EST has been verified by BLAST. A typical library may have 10,000 to 30,000 clones in twenty to thirty 384-well plates.

Multiply the Possibilities with ReplicatedDNA Clone Plates

In This Issue

p. 1Clone PlatesStem Cell Pluripotency

p. 2What’s New: Extremophiles

p. 5Stem Cell Markers

p. 7Tech Qs: 293 Cells

p. 8MALDI and Staph

p. 10Preserving Animal Cells

p. 12Searching for Clones

continues on page 4 ww

Volume 24 Numb er 2

Pluripotent stem cells, including embry-onic stem (ES) cells, embryonal car-

cinoma (EC) cells, and embryonic germ (EG) cells, exhibit unique properties that have been exploited for stem cell deriva-tion, purification, and analysis (1,2,3). The transcription factor protein Oct-3/4 and enzymes such as phosphatases have been investigated as potential markers for identi-fying and distinguishing differentiation in embryonic stem cell populations in vitro. Immunocytochemical methods including the Gomori technique, azo dye methods and BCIP/NBT have been employed to

detect endogenous alkaline phospha-tase activity in cells (4,5).

Using the parental mouse embryonic stem cell line R1 and its subclone R1/E, we demonstrated a novel dual-color fluorescence-based protocol for detecting and comparing Oct-3/4 expression and phosphatase activity simultaneously in embryonic stem cells. The results were then compared to our germline transmission data for these ES lines, assessing the feasibility

Fluorescence-Based Analysis of Embryonic Stem Cell Pluripotency

continues on page 3 ww

T.W. Plaia, R. Josephson, H. Mitchell, A. Toumadje, T. Tavakoli, W. Xu, and J.M. Auerbach, Stem Cell Center at ATCC; J. Morgan, The Jackson Laboratory.*

New ExtremophilesAn extreme psychrophile, Colwellia psychr-erythraea was isolated from Arctic shelf sediments off the northeast coast of Green-land. It grows at 4°C and has extracellular enzyme activity (Environ. Microbiol. 2(4): 383-388, 2000). The genomic DNA from this organism is also available as BAA-681D. ATCC No. Unit PriceBAA-681 1 vial $150

Echinamoeba thermarum is a newly described thermophilic protozoan isolated from a hot spring in Yellowstone National Park (Extremophiles 7(4):267-274, 2003). This fascinating eukaryote prefers to grow at 50°C in medium of pH 6. The OSB1 strain of E. thermarum is exclusively heat-adapted and does not grow or remain active at ambient temperatures.

This strain is part of ATCC’s National Parks Service Special Collection. Visit our Web site at www.atcc.org to search our Protistology Collection or to learn more about National Park Service strains.

ATCC No. Unit PricePRA-13 1 vial $145

More Extremophile InfoATCC currently houses a wide selection of extremophilic archaea and bacteria iso-lated from a variety of environmental sources. Download our new brochure at www.atcc.org/pdf/eep.pdf. And when you’ve found the organism that meets the needs of your research, don’t forget the needs of your organism. ATCC offers both a

This newsletter is published by ATCC and is distributed free of charge upon request. Direct all correspondence to P.O. Box 1549, Manassas, VA, 20108, or e-mail news@atcc.org. Photocopies may be made for personal or internal use without charge. This consent does not extend to copying for general distribution, promotion, creating new works, or resale. © 2004, ATCC. ISSN 1088-2103 ATCC is a registered trademark of the American Type Culture Collection.

The products described in ATCC Connection are intended for research purposes only.They are not intended for use in humans.

2 ATCC Connection

Vitamin Supplement (catalog no. MD-VS) and a Trace Mineral Supplement (catalog no. MD-TMS) to meet the demands of a growing extremophile culture.

Mouse-adapted ProtozoanThe Peabody strain of Babesia microti is a human isolate that has been adapted to grow in mice (J. Parasitol. 65(3): 430-433, 1979).

ATCC No. Unit PricePRA-99 1 vial $145

Urease-negative HelicobacterHelicobacter rodentium was isolated from a laboratory mouse. It is urease negative and one of only two Helicobacter species with unsheathed flagella (Int. J. Syst. Bac-teriol. 47(3): 627-634, 1997).

ATCC No. Unit Price700285 1 vial $190700286 1 vial $190

Cell Immortalization Products ATCC has been granted a license from Geron Corporation to create and distrib-ute products related to human telomer-ase reverse transcriptase (hTERT).

Several product lines are planned: • Immortalized cell lines and hTERT plasmids created by Geron. • Immortalized ATCC cell lines. • Kits and support products.

The first products will be available soon. If you would like to receive e-mail

updates regarding the status of hTERT products from ATCC, visit our home page at www.atcc.org. Look for the sign-up link and check the box for receiving cell immortalization updates.

The Means–and the Genes–to an End

The induction of apoptosis generally fol-lows one of two related biochemical pathways. The extrinsic path-way occurs when the cell is induced to die by activation of death receptors that are members of the tumor necrosis factor (TNF) superfamily of pro-teins. The intrinsic pathway occurs when the cell is deprived of survival factors such as cytokines and growth factors or exposed to external toxins, intracellular damage, or stress signals.

ATCC has developed schematics of these two pathways which depict the genes involved as well as the biochemical and morphological changes that accompany apoptosis. You can follow links to NCBI gene data and learn about clone availabil-ity in ATCC’s catalog. Apoptosis detection kits and related cell lines are also noted when appropriate.

To get to the pathways follow the link from our home page at www.atcc.org.

What’s new

3 ATCC Connection

Plates can be purchased individually or in sets. Volume dis-counts are available. The plate number (as found on the IMAGE Web site) serves as the ATCC catalog number. To order call

Xenopus Clone Plates from the IMAGE Consortium

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)Full length MGC IRBG 96 $288 Full length MGC IRBH 96 $288 Full length MGC IRBN 96 $288 Putative MGC IRAK 384 $700 Putative MGC IRAL 384 $700 EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Zebrafish Clone Plates from the IMAGE Consortium

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate) Full length MGC IRBO 96 $288 Putative MGC IRAK 384 $700 Putative MGC IRAL 384 $700 EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Rhesus Monkey Clone Plates from the IMAGE Consortium

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Soybean Clone Plates

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)EST Soybean EST SYBN 384 $600 Project

NIA/NIH Mouse Embryo Clone Plates

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)EST 15K set H3001- 96 $288 H3159 EST 7.4K set H4001- 96 $288 H4079

Clone Platesvvcontinued from p. 1

800-638-6597 or e-mail clones@atcc.org.

Human Clone Plates from the IMAGE Consortium Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate) Full length MGC IRAT 96 $400 Full length MGC IRAU 96 $400 Putative PCR rescue genes IRBK 96 $288 Putative PCR rescue genes IRBR 96 $288 Putative PCR rescue genes IRBU 96 $288 Putative G-related proteins IRBF 96 $288 (GPCR) Putative G-related proteins IRBI 96 $288 (GPCR) Putative MGC IRAK 384 $700 Putative MGC IRAL 384 $700 EST Glioma related IRAX 96 $288 EST Lymphochip IRAY 96 $288 EST Lymphochip IRAZ 96 $288 EST Lymphochip IRBA 96 $288 EST Lymphochip IRBB 96 $288 EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Mouse Clone Plates from the IMAGE Consortium

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)Full length MGC IRAV 96 $400 Full length MGC IRAW 96 $400 Putative MGC IRAK 384 $700 Putative MGC IRAL 384 $700 EST Lymphochip IRAP 96 $288 EST Pancreas IRBD 96 $288 EST Pancreas IRBE 96 $288 EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Rat Clone Plates from the IMAGE Consortium

Clone Type Clone Collection Plate Plate List Price Prefix Format (per plate)Full length MGC IRBP 96 $400 Full length MGC IRBQ 96 $400 Putative MGC IRAK 384 $700 Putative MGC IRAL 384 $700 EST Univ. of Iowa IRAD 96 $288 EST IMAGE LLAM 384 $130 EST IMAGE LLCM 384 $130 EST IMAGE LLKM 384 $130

Product Focus

4 ATCC Connection

of complete in vitro qualification of ES pluripotency.

Materials and methods

Embryonic stem cells were grown on mouse embryonic fibroblasts for 3 to 4 days to obtain colonies. Cells were fixed with 4% paraformaldehyde, permeabi-lized with 0.2% Tween-20 (ICI Ameri-cas), and blocked with 3% normal goat serum. Monoclonal anti-Oct-3/4 antibody was added and allowed to incubate for one hour, followed by Texas Red goat anti-mouse IgG (H+L) antibody (Molecu-lar Probes) for 1 hour. A 1:20 dilution of ELF 97 phosphatase substrate (Molecu-lar Probes) was added and the reaction was monitored under an epifluorescence microscope. The reaction was stopped and cells were washed with PBS before mounting.

Results

The enzyme-linked fluorescence (ELF) reagent caused surface alkaline phos-phatases (AP) to glow yellow-green under fluorescent light at 530 nm (Figure

1). Oct-3/4 was detected using Texas Red at 615 nm, yielding red fluorescence. Both markers can be visualized simul-taneously, producing dual-color mottled pluripotent stem cells (Figure 2).

Conclusions

The dual-color assay can be utilized to assess overall in vitro embryonic stem cell pluripotency. We were able to detect and visualize intracellular (Oct-3/4) and cell surface (AP) embryonic stem cell marker expression. Qualitative analysis of these markers in both R1 and R1/E germ-line competent embryonic stem cells reveals a slightly higher detection of AP activity in the R1 ES cells, which may cor-relate to its increased level of germline transmission.

References

1. Shamblott MJ et. al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95(23): 13726-13731, 1998.

2. Nagy A et. al. Manipulating the mouse embryo: a laboratory manual, 3rd ed. Cold Spring Harbor Press: Cold Spring Harbor, New York; 2002.

3. Nagy A et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90(18): 8424-8428, 1993.

4. Telford WG et al. Detection of endogenous alka-line phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phospha-tase substrate. Cytometry 37(4): 314-319, 1999.

5. Cox WG et al. A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity. J. Histochem. Cyto-chem. 47(11): 1443-1456, 1999.

This project is supported by NCRR (P40-RR15452). It was presented as a poster at the 2004 meeting of the International Society for Stem Cell Research and highlighted in Biophotonics International (Aug. 2004).

*J. Morgan is currently with the Washington Depart-ment of Fish and Wildlife

A

B

C

D

E

F

Figure 1. Analysis of alkaline phosphatase activity in ES cells. The green fluorescence of ELF 97 reagent is overlaid on brightfield images in R1 cells.

Figure 2. Analysis of alkaline phosphatase (AP) activity and Oct-3/4 expression. Oct-3/4 expression was detected with Texas Red in A) R1 and B) R1/E cells. Endogenous AP activity was detected by ELF 97 reagent in C) R1 and D) R1E cells. Markers are visualized simultaneously in E) R1 and F) R1/E cells.

Stem Cell Pluripotency vvcontinued from p. 1

New Products from the Stem Cell Center

Stem CellsMouse embryonic stem cell CE-3

(SCRC-1039)

Feeder Layer CellsIrradiated human fibroblasts (SCRC-1041.1)AFT024, irradiated (SCRC-1007.1)MitC-treated human fibroblasts

(SCRC-1041.2)MitC-treated mouse fibroblasts

(SCRC-1046.2)

Media, Serum, and ReagentsES-Qualified DMEM (SCRR-2010)L-Alanyl-L-Glutamine, 200 mM (30-2115)MEM Nonessential Amino Acid Solution,

100x (30-2116)PBS Without Calcium or Magnesium

(SCRR-2201)ES-Qualified Fetal Bovine Serum (SCRR-

30-2020)

Product Focus

5 ATCC Connection

Undifferentiated ES and Differentiated EB Markers of R1 and R1/E CellsA. Toumadje, T. Tavakoli, T.W. Plaia, W. Xu, H. Mitchell, R. Josephson, J.M. Auerbach, Stem Cell Center at ATCC; E. Cedrone, Cell Biology Program, ATCC.

continues on page 6 ww

Figure 1. Separation of feeder cells (green) from embryonic stem cells (blue) based on their forward scattered light (size) and side scattered light (com-plexity). The density plot displays forward (X) versus side scattered (Y) light properties. The histogram depicts the fluorescence intensity (X) versus cell number (Y).

Figure 2. Expression of Oct-3/4 in R1/E and R1 cells. Flow cytometry results show high expression in undifferentiated stem cells (A and C) and low in differentiated embryoid bodies (B and D).

In vitro characterization of stem cells is a rapid and convenient approach for mon-itoring and predicting developmental pluripotency. In this study we used flow cytometry to characterize two mouse embryonic stem (ES) cell lines, R1 and the R1/E subclone (1), following their tran-sition in vitro from the undifferentiated state to their differentiation into spher-ical multicellular aggregates known as embryoid bodies or EB (2).

We monitored this transition based on the differential immunophenotype stain-ing capacity of the cells to a panel of anti-bodies. The panel consists of pluripotent stem cell markers Oct-3/4 (a stem cell-specific transcription factor) and SSEA-1 (a stage-specific embryonic antigen), and the differentiation marker cytokeratin endo-A antigen TROMA-1 (3,4).

Materials and methods

In order to maintain the undifferentiated, pluripotent state of ES cells, both R1 and R1/E were cocultured with mitomycin-treated CF-1 mouse embryonic fibroblast cells (ATCC SCRC-1040.2 ) in ES-DMEM medium (catalog no. SCRR-2010) supple-mented with L-alanyl-L-glutamine (cat-alog no. SCRR-30-2115), non-essential

amino acids (catalog no. 30-2116), β-mercaptoethanol (Invitrogen), mouse leukemia inhibitory factor (LIF, Chemi-con), and ES-qualified fetal bovine serum (catalog no. SCRR-30-2020). ES cells were cultured without feeder cells in complete ES medium lacking LIF to initiate EB for-mation.

For flow cytometric single-cell prepara-tion and antibody staining, cells were harvested using 0.25% trypsin/0.53 mM EDTA solution (catalog no. 30-2101) in calcium- and magnesium-free PBS (cata-log no. SCRR-2201), fixed in 1% parafor-maldehyde, and permeabilized in 0.5%

saponin for intracellular staining. The purified monoclonal antibodies used were anti-Oct-3/4 (Transduction Lab), anti-SSEA-1 (Chemicon), and anti-TROMA-1 (Developmental Studies Hybridoma Bank). FITC or Alexa-labeled secondary antibodies against host pri-mary antibodies were used (Molecular Probes).

Flow cytometry was performed on a BD FACSCalibur (BD Biosciences Immuno-cytometry Systems) equipped with an air-cooled 488-nm argon laser and a 635- nm red diode laser. Ten thousand events were acquired and analyzed by either

Table 1. Percent of R1 and R1/E cells positive for Oct-3/4, SSEA-1, and TROMA-1 in undifferentiated cells (Day 0) to embryoid bodies (Day 14).

R1 R1/E Day 0 Day 14 Day 0 Day 14Oct-3/4 92% 1% 91% 1%SSEA-1 92% 27% 98% 22%TROMA-1 4% 33% 5% 52%

A B

C D

Focus

6 ATCC Connection

vvcontinued from p. 5

BD Cellquest software (Becton, Dickin-son and Company) or WinList software (Verity Software House, Inc). Feeder cells were gated out of the analysis based on their forward and side scattered light (size) and their autofluorescence proper-ties.

Results

Gating of feeder cells from ES cells. In general, feeder cells showed higher forward and side scatter than ES cells. Additionally, feeder cells emitted a more intense autofluorescent background than ES cells (Figure 1). We verified this phenomenon by analyzing feeder cells alone without ES cells (data not shown).

Analysis of stem cell markers during differentiation. By excluding the feeder cells from analysis using the scattered light size gating, we were able to perform immunophenotype analysis of stem cells against stem cell markers Oct-3/4 and SSEA-1 and differentiation marker TROMA-1. We showed that the percentage of positive cells (Table 1) and the intensity of expression per cell declined during in vitro EB development for Oct-3/4 and SSEA-1 (Figures 2 and 3).

Conversely, TROMA-1 was upregulated during EB formation (Figure 4). The dim staining of undifferentiated ES cells for TROMA-1 (Figure 4A) is not visible in fluo-rescence microscopy. It is important to

view cells using fluorescence microscopy in parallel with flow cytometry in order to monitor cell integrity and staining dis-tribution in EB aggregates and to dis-tinguish the positive region from the background (rectangular region in Figure 4A).

The pluripotent R1 ES cell line was established in 1991. It is reported that prolonged in vitro culture (passage 14) diminishes the totipotency of this ES line (2). However, the R1/E subclone of R1 is claimed to maintain its germline compe-tency much longer than the parental R1 line. In this report we used R1 and R1/E at passage 16 and 11, respectively. The data show that these two ES lines do not differ in their expression of markers for undif-ferentiated stem cells and differentiated

EBs.

This project is supported by NCRR (P40-RR15452) and was presented as a poster at the 2004 meeting of the International Society for Stem Cell Research.

References

1. Nagy A et al. Derivation of completely cell-cul-ture derived mice from early-passage embry-onic stem cells. Proc. Natl. Acad. Sci. USA 90(18): 8424-8428, 1993.

2. Keller GM. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7(6): 862-869, 1995.

3. Okamoto K et al. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60(3): 461-472, 1990.

4. Solter D and Knowles BB. Monoclonal anti-body defining a stage-specific mouse embry-onic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75(11): 5565-5569, 1978.

Figure 3. Expression of SSEA-1 in R1/E cells. Flow cytometry results show high expression in undifferenti-ated stem cells (A) and low in differentiated embryoid bodies (B).

Figure 4. Expression of TROMA-1 in R1 cells. Flow cytometry results show low expression in undifferenti-ated stem cells (A) and high in 14-day-old differentiated embryoid bodies (B). The heterogenous popula-tion of embryoid bodies causes the broad histogram.

Sign Up!

You can receive product news and technical tips from ATCC absolutely free. Visit our home page at www.atcc.org and look for the sign-up link.

A B

A B

Focus

7 ATCC Connection

TechQs are intended for informational purposes only. ATCC is not responsible for results obtained from the use of this information.

Tech Qs

Why are my 293 cells detaching? Is this normal?

These cells are known for being problem-atic in terms of detachment and clumping. If flasks are left at room temperature, 293 cells have the tendency to spontaneously detach. Likewise, the cells have also been known to detach at confluence. For this reason, we recommend subculturing at 70 to 80% confluence.

If your cells have detached and are clump-ing in the medium, it will take some patience and manipulation to coax the cells into lying flat again. Start by gently pipet-ting against the side of the flask to break up clusters into a single-cell suspension. Use a minimal amount of medium to reseed the culture (12 ml for a T-75 flask), and allow the cells to incubate undisturbed for about 24 to 48 hours.

After incubation you may want to avoid complete fluid changes. Instead try adding small amounts of medium to the culture vessel so the cells are not dislodged from the surface of the flask during fluid renewal.

Keep in mind, however, that the cells may still clump after reattaching to the flask.

When the cells are ready for subculture, try breaking up clusters during dissociation in the trypsin/EDTA solution (catalog no. 30-2101) before neutralizing with medium containing serum. However, take care to perform this procedure quickly so as not to over-trypsinize the cells. Again, seed the cells to a new flask with a minimal volume of medium, adding medium every 2 to 3 days instead of performing complete fluid changes.

ATCC rarely has problems with the attach-ment of 293 cells. For optimal recovery and maintenance we recommend using ATCC medium, serum, and reagents. ATCC CRL-1573 293 cells are propagated in 90% Eagle’s Minimum Essential Medium (catalog no. 30-2003) and 10% heat-inacti-vated horse serum (catalog no. 30-2041). ATCC’s medium formulation contains 2 mM L-glutamine, Earle’s balanced salt solution, 1.5 g/liter sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate.

The following procedure can be followed to heat-inactivate serum:

1. Remove the serum from frozen storage and place it overnight in a refrigerator

at 2 to 6°C. Transfer the bottles to a 37°C water bath. Agitate the bottles from time to time in order to mix the solutes that tend to concentrate at the bottom of the bottle. Do not keep the serum at 37°C any longer than necessary to completely thaw it.

2. Preheat water bath to 56°C. There must be sufficient water to immerse the bottle above the level of serum.

3. Mix thawed serum by gentle inversion and place serum bottle in the 56°C water bath. The temperature of the water bath will drop.

4. When the temperature of the water bath reaches 56°C again, continue to heat for an additional 30 minutes. Mix gently every 5 minutes to ensure uniform heating.

5. Remove serum from water bath and cool. Place at -70°C if possible, otherwise at -20°C.

More cell culture tips can be found in the Frequently Asked Questions on our Web site. Go to the home page at www.atcc.org, and choose the Tech Support link at the top.

Focus

8 ATCC Connection

Colony variants are not uncommon among Staphylococcus aureus strains (1). For example, ATCC 43300 S. aureus exhibits both beta-hemolytic and non-hemolytic colony types when grown on trypticase soy agar with 5% defibrinated sheep blood at 37oC for 24 hours. These colony types remain distinct when sub-cultured onto solid medium.

This strain is also a low-expression-class methicillin-resistant S. aureus, or MRSA (2). Its heterogeneous expression of oxa-cillin resistance, which allows the clinician to test a heteroresistant population (3), makes it a common control strain in anti-biotic testing protocols.

We investigated the two colony types of S. aureus using a variety of methods: traditional biochemicals, ribotyping, 16S rDNA sequencing, and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). MALDI-TOF analyzes the surface components of cells and produces a spectrum that is unique to each organism (4).

To determine whether dif-ferences in the variants is limited to hemolysis, we selected additional staphy-lococci based on coagulase reaction, antibiotic resis-tance, and colony morphol-ogy for analysis by MALDI-TOF. Other organisms such as Escherichia coli and Strep-tococcus dysgalactiae were also included because of their colony variation and hemolytic reaction.

Materials and methods

All cultures were obtained from the Bac-teriology Collection at ATCC (Table 1). Strains were grown on tryptic soy agar (TSA) with 5% sheep blood or brain heart infusion broth for 24 hours. Each hemolytic variant was identified using an API Staph strip (bioMerieux). Ribotyping analysis was performed using a Ribo-Printer (Qualicon) according to manu-

facturer’s instructions. Samples for fatty acid methyl ester analy-sis (FAME) were grown on TSA with 5% sheep blood at 37°C in ambient air for 24 hours. Cells were harvested and extracted using the MIDI Inc. protocol. Samples were then analyzed on a HP6890 GC system (Agilent Technologies). Identifica-tions were derived using the Sherlock Microbial Identification System (MIDI Inc.). Universal primers were used to amplify a portion of the 16S region of each isolate for rDNA analysis. This region was then sequenced using CEQ 8000 (Beckman Coulter) and analyzed using the Ribo-somal Database Project II Web site (5). All samples were analyzed using a Micro-mass MALDI-TOF-MS (MALDI-L, Waters Corp.) operated in linear mode. The system comes with MassLynx 3.5 soft-ware for instrument control and Microbe-Lynx software for data analysis.

Results

Characterization of the two colony types

MALDI-TOF Analysis of Hemolytic Variants of Staphylococcus aureus Kim Greth, Paul Krader, and Jane Tang, ATCC

Table 1. Bacterial strains used in this study. Additional strains were also tested with results similar to those presented here.

ATCC No. Organism Name Hemolysis Coagulase43300 Staphylococcus aureus subsp. aureus Beta + 43300 Staphylococcus aureus subsp. aureus None +BAA-41 Staphylococcus aureus subsp. aureus Beta +BAA-41 Staphylococcus aureus subsp. aureus None + 700788 Staphylococcus aureus subsp. aureus Beta +BAA-39 Staphylococcus aureus subsp. aureus None +700373 Staphylococcus lutrae Beta +49051 Staphylococcus intermedius Beta +49052 Staphylococcus intermedius None -43809 Staphylococcus lugdunensis Weak beta -49576 Staphylococcus lugdunensis None -43808 Staphylococcus shleiferi subsp. schleiferi Beta -49680 Staphylococcus shleiferi subsp. coagulans None -25922 Escherichia coli Slight beta NA27957 Streptococcus dysgalactiae group C None NABAA-337 Streptococcus dysgalactiae group A Beta NA

Figure 1. Riboprint results from the beta-hemolytic (B) colony variant and the non-beta-hemolytic (NB) variant. The genetic fingerprints for these two colony variations is essentially the same with a similarity index of 0.98.

Focus

9 ATCC Connection

using phenotypic tests, fatty acid profiles, DNA fingerprinting (Figure 1) and 16S rDNA sequencing confirm they are the same organism. Their antimicrobial resis-tance patterns were identical. However, each variant produced a unique spec-trum with MALDI-TOF (Figure 2).

These spectra have shown to be consis-tent with hemolytic and nonhemolytic variants in all S. aureus tested (Figure 3). In other organisms which also exhibit differ-ent hemolytic phenotypes such as Esche-richia coli and Streptococcus dysgalactiae, the spectra seem to be independent of hemolysis.

Discussion

The results showed that beta-hemolytic staphylococci were unique in producing a membrane component between 2 and 3.5 kDa in size which was not evident in nonhemolytic staphylococci, E. coli,

or S. dysgalactiae. This suggests that a major protein associated with hemolysis is expressed on the cell surface of staphylococci which the MALDI-TOF technol-ogy was capable of detecting.

Acknowledgement

We would like to thank Jason Cooper of ATCC for contributing the 16S rDNA sequences of these organisms.

This study was presented as a poster at the 2004 annual meeting of the Ameri-can Society for Microbiology.

References

1. Bannerman TL. Staphylococcus, Micro-coccus, and other catalase-positive cocci that grow aerobically. In: Murray PR et al (eds.). Manual of Clinical Microbiology. Washington DC: ASM Press; 2003: pp. 384-404.

2. Mackenzie AM et al. Evidence that the National Committee for Clinical Labo-ratory Standards disk test is less sen-

sitive than the screen plate for detection of low-expres-sion-class methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 33(7): 1909-1911, 1995.

3. Swenson JM et al. Optimal inoculation methods and quality control for the NCCLS oxacillin agar screen test for detection of oxacillin resistance in Staphylococcus aureus. J. Clin. Micro-biol. 39(10): 3781-3784, 2001.

4. Walker J et al. Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility. J. Microbiol. Methods 48(2-3):117-126, 2002.

5. Ribosomal Database Project II. http://rdp.cme.msu.edu/index.jsp.

Figure 2. MALDI-TOF spectra of the 12 runs used to obtain the final spectrum for each isolate. The combined spectrum is in green. Patterns shown are for a mix of the beta- and non-beta-hemolytic colony variants of ATCC 43300 Staphylococ-cus aureus (top), the beta-hemolytic variant only, and the non-beta-hemolytic variant only. The mixed pattern appears to be a combination of the beta and non-beta-hemolytic patterns.

Figure 3. Spectra of Staphylococcus strains. The spectra of beta-hemolytic strains are shown in blue and those of non-beta-hemo-lytic strains are shown in red. The difference in spectra is not dependent on coagulase reaction but does appear to be consis-tent with hemolytic reaction.

Focus

Most cell cultures can be stored for many years, if not indefinitely, at temper-atures below -130°C (cryopreservation). The many advantages of cryopreserva-tion far outweigh the required invest-ment in equipment and reagents, such as generating safety stocks, preserving cells with finite population doublings, protect-ing against phenotypic drift, and creating standard reagents to be used for a series of experiments.

Overview

As the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases. Intracellular ice can be minimized if water within the cell is allowed to escape by osmosis during the cooling process. A slow cooling rate, gen-erally -1°C per minute, facilitates this pro-cess. However, as the cells lose water, they shrink in size and will quickly lose viability if they go beyond a minimum volume. The addition of cryoprotectant agents such as glycerol or dimethylsulf-oxide (DMSO) will mitigate these effects.

The standard procedure for cryopreser-vation is to freeze cells slowly until they reach a temperature below -70°C in medium that includes a cryoprotectant. Then, transfer the vials to a liquid nitro-gen freezer to maintain them at tempera-tures below -130°C.

The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water bath at 37°C, removed from the freeze-medium by gentle centrifugation and/or diluted with growth medium, and seeded in a culture vessel in complete growth medium.

There are numerous factors which affect the viability of recovered cells. Modify the procedure for each cell line to attain opti-mal cell viability upon recovery. Some of the critical parameters for optimization include the composition of the freeze medium, the growth phase of the culture, the stage of the cell in the cell cycle and the number and concentration of cells within the freezing solution.

Freeze medium

Glycerol and DMSO at 5 to 10% are the most common cryoprotectant agents. While DMSO can be toxic to cells, it penetrates them much faster than glyc-erol and yields more reproducible results. Unfortunately, DMSO can cause some cells to differentiate (e.g., HL-60 promy-eloblast cells) and may be too toxic for some cells (e.g., HBE4-E6/E7 lung epi-thelial cells). Glycerol should be used in these instances. Glycerol can be sterilized by autoclaving whereas DMSO must be sterilized by filtration. Care should be used when handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it.

Use only reagent-grade (or better such as cell culture-grade) DMSO or glycerol. Store both in aliquots protected from light. ATCC offers cell culture grade DMSO (catalog no. 4-X) that has been thor-oughly tested to ensure it is nontoxic. For cells grown in serum-free medium, adding 50% conditioned medium (serum-free medium in which the cells were grown for 24 hours) to both the cell freezing and the recovery medium may improve recovery and survival. The addition of 10 to 20% cell culture-grade bovine serum albumin to serum-free freezing medium may also increase post-freeze survival.

Other variations on freeze medium for-mulations include high (up to 90%) con-centrations of serum which presumably supplies some cryoprotection as well as additional growth factors; use of a bal-anced salt solution designed for hypo-thermal conditions in place of medium designed for 37°C incubation; and the addition of apoptotic inhibitors which may prevent delayed onset cell death fol-lowing recovery (1). Optimum formula-tions for individual cell lines need to be determined empirically.

Equipment

Cryopreservation vialsThere are two materials to choose from

for cryopreservation vials: glass or plastic. Glass vials are more difficult to work with; they need to be sterilized before use, they do not come with labels, they need to be sealed with a hot flame to melt the glass neck, they will shatter if dropped, and they can be difficult to open. How-ever, they are preferred for long-term storage (many years) of valuable cultures and are considered fail-safe once prop-erly sealed. ATCC uses glass vials for the storage of seed stocks but plastic vials for storage of distribution stocks of our cell lines.

Plastic vials come in two varieties: those with an internal thread and silicone gasket and those with an external thread. The internal-thread version was the first commercially available, but has some disadvantages over the external-thread version. For example, while the silicone gasket provides an excellent seal, it needs to be tightened just right; too tight or too loose and the vial will leak.

Controlled-rate freezing chambersThere are several means to achieve a cooling rate of -1°C per minute. The best is with a computer controlled, program-mable electronic freezing unit (e.g., Cryo- Med Freeze) which rigorously maintains this rate of cooling. This is the method used exclusively at ATCC. Such equip-ment is expensive and absolutely neces-sary for only the most sensitive cells. A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a -70°C (or lower) mechanical freezer. There are several commercially available freezing chambers which achieve a cooling rate very close to the ideal -1°C per minute (Mr. Frosty, Nalgene Catalog # 5100-0001; or StrataCooler, Stratagene Catalog # 400005). Alternately, the vials can be placed into a Styrofoam box with 15-mm (3/4 inch) thick walls and 1-liter capacity packed with paper, cotton wool, or Styro-foam peanuts for insulation.

Storage

The ultralow temperatures (below -130°C) required for long-term storage

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can be maintained by specialized electric freezers or more commonly by liquid nitrogen freezers. There are two basic types of liquid nitrogen storage systems: immersing vials in the liquid and holding vials in the vapor phase above the liquid. The liquid-phase system holds more nitrogen and thus requires less mainte-nance. However, there is always a chance that some liquid will enter improperly sealed vials which may explode when retrieved. For this reason ATCC strongly recommends storage in vapor-phase sys-tems.

Vapor-phase systems create a vertical temperature gradient within the con-tainer. The temperature in the liquid nitrogen at the bottom will be -196°C, whereas the temperature at the top will vary depending upon the amount of liquid nitrogen at the bottom as well as the amount of time the container is opened. To ensure safe storage of your cells, be sure to keep enough liquid nitro-gen in the container so that the tempera-ture at the top (just above the storage level) is -130°C or lower. All storage sys-tems should be equipped with tempera-ture alarms.

Cryopreservation procedure

The procedure below will work for most cell cultures and should be modified as needed. Freeze medium formulations for all ATCC cell lines are provided on the product information sheet. Harvest cells in exponential growth.

1. Check your cell culture for contamina-tion from bacteria, fungi, mycoplasma, and viruses immediately before cryo-preservation. In most cases, the results of the contamination screen will be available some time after the cultures are cryopreserved. If contamination is confirmed, destroy the frozen material.

2. Prepare a freeze medium consisting of complete growth medium and 5% DMSO. Do not add undiluted DMSO to a cell suspension as dissolution of DMSO in aqueous solutions gives off heat.

3. Collect cells by gentle centrifugation

(10 min at 125 x g) and resuspend them in the freeze medium at a con-centration of 1 x 106 to 5 x 106 viable cells/ml. Continue to maintain the cells in culture until the viability of the recovered cells is confirmed (see Step 9).

4. Label the appropriate number of cryo-vials with the name of the cell line and the date. Then add 1 to 1.8 ml of the cell suspension to each of the vials (depending upon the volume of the vial) and seal.

5. Allow cells to equilibrate in the freeze medium at room temperature for a minimum of 15 minutes but no longer than 60. This time is usually taken up in dispensing aliquots of the cell suspen-sion into the vials. After 60 minutes, cell viability may decline due to the DMSO.

6. Place the vials into a controlled-rate freeze chamber and place the cham-ber in a -70°C (or colder) mechanical freezer for at least 24 hours. Alter-nately, use a programmable freezer unit set to cool the vials at -1°C per minute until a temperature below -70°C is achieved.

7. Quickly transfer the vials to a liquid nitrogen or -130°C freezer. Frozen material will warm up at a rate of 10°C per minute and cells will deteriorate rapidly if held above -50°C for any length of time.

8. Record the location and details of the freeze.

9. After 24 hours at -130°C, remove one vial, restore the cells in culture, and determine their viability and sterility.

Recovery of cryopreserved cells

The cell solution in the frozen vial needs to be warmed as rapidly as possible and then immediately combined with com-plete culture medium and seeded into an appropriate flask. While cells grown in monolayers can be recovered from cryopreservation in multiwell plates, the results are not as consistent as with flasks.

Some cell lines, such as hybridomas cul-tures, take several days before they fully recover from cryopreservation. Indeed,

some hybridomas appear dead the first day in culture and will generate a lot of cellular debris. Viability for most cells declines and reaches a nadir at 24 hours post-thaw. Most, if not all, of this decline appears to be due to apoptosis (as opposed to necrosis) induced by the stress of the cryopreservation process (2). After this time point, cells begin to recover and enter exponential growth.

1. Remove the vial from the liquid nitro-gen freezer and thaw by gentle agita-tion in a 37°C water bath (or a bath set at the normal growth temperature for that cell line). Thaw rapidly (approxi-mately 2 minutes).

2. Remove the vial from the water bath as soon as the contents are thawed and decontaminate it by dipping in or spraying with 70% ethanol. Carry out all of the operations from this point on in a laminar flow tissue culture hood under strict aseptic conditions.

3. Prepare a culture vessel (such as a T-75 flask) so that it contains at least 15 ml of the appropriate culture medium equilibrated for temperature and pH.

4. Unscrew the top of the vial and remove the cryoprotectant agent by gentle centrifugation (10 minutes at 125 x g), discard the supernatant, and resus-pend the cells in 1 or 2 ml of complete growth medium. Transfer the cell sus-pension into the medium in the culture vessel and mix thoroughly.

5. Examine the cultures after 24 hours and subculture as needed.

This procedure is available in the Tech Support section of ATCC’s Web site as Technical Bulletin No. 3.

References

1. Baust JM et al. Cell Preservation Technology1: 17-31, 2002.

2. Baust JM et al. Cell Preservation Technology 1: 63-80, 2002.

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