INTRODUCTION Microscopic analysis of live cells has historically been

performed by growing cells on cover slips and inverting them

onto glass slides to place under a microscope. Because this

process is often messy and inefficient, modern cell biology

laboratories often use chambered slides to avoid transferring

cells from culture vessels to microscope slides. Chambered

slides are especially convenient for immunocytochemistry, in

which fluorescent stains are used to visualize cell markers and

substructures.

The new Millicell EZ slide enables cell biologists to culture,

treat, fix, stain, and view cells all directly on a microscope slide.

Improved Analysis of Cell Lines and Neural Stem Cells with Millicell® EZ slides

Application Note

Figure 1. The EZ slide’s unique design enables easy well removal. The geometry of the Millicell EZ slide is optimized to prevent cross contamination, and its cover has been designed for easy visualization and for convenient stacking of multiple devices in a laminar flow hood or incubator.

4-well 8-well

Break-away Tabs

Slide Holder

Slide

Wells

Lid

This new product has several unique advantages over

currently available chambered slides.

• Millicell EZ slides are chemically compatible with most fixing

and staining reagents, eliminating the need to remove

chambers prior to analysis.

• Breakaway snap closure eliminates the risk of breaking

slides when removing wells (Figure 1).

• Wells detach easily from the slides without glue

or gasket residue.

• No tools are required to detach wells from the slide.

2

MATERIALS AND METHODS

Immunocytochemistry of NIH 3T3 cellsNIH 3T3 cells were seeded at 40,000 cells/cm2, with 1.7 mL/

well culture medium in Brand B 4-chamber glass culture

slides or Millicell 4-well glass EZ slides. Cells were incubated

for 24 h at 37 °C, 6% CO2, and 95% relative humidity (RH).

Cells were then fixed with 4% paraformaldehyde and stained

with Hoechst nuclear dye, Alexa Fluor® 546-conjugated

phalloidin (Life Technologies), and SYTO®13 green fluorescent

nucleic acid stain (Life Technologies).

Immunochemistry of CHO-K1 cells CHO-K1 cells were seeded at 40,000 cells/cm2, in Brand B

glass culture slides or Millicell glass EZ slides. Cells were

cultured in 1.7 mL/well culture medium (for 4-chambered

slides) or 0.4 mL/well culture medium (for 8-chambered

slides). Cells were incubated for 24 h at 37 °C, 6% CO2, and

95% relative humidity (RH). Cells were then fixed with 4%

paraformaldehyde and stained with Hoechst nuclear dye,

Alexa Fluor 546-conjugated phalloidin (Life Technologies), and

SYTO13 green fluorescent nucleic acid stain (Life

Technologies).

Immunocytochemistry of ReNcell® CX neural stem cellsUndifferentiated ReNcell CX neural stem cells (NSC) (Millipore)

were cultured in ReNcell NSC expansion medium (Millipore)

containing 20ng/mL FGF-2 and 20 ng/mL EGF. ReNcell CX

cells were plated in Millicell 8-well glass EZ slides or Brand B

8-well glass slides precoated with 20 mg/mL laminin

(Millipore) at 50,000 cells/well in 0.5 mL/well medium and

incubated at 37 °C, 5% CO2. The next day, cells were fixed

with 4% PFA for 45 minutes and incubated in 3% goat serum

and 0.1% Triton X-100. Samples were incubated for 2h at RT

with anti-nestin (Millipore) and anti-Sox2 (Millipore) primary

antibodies. Cells were then incubated with secondary

antibodies (goat anti-mouse and goat anti-rabbit) and

Hoechst nuclear stain.

RESULTS AND DISCUSSIONNIH 3T3 cells showed no difference in qualitative

proliferation and cell morphology between Millicell EZ slides

and Brand B chambered slides, as evidenced by conserved

organization patterns of actin-based structures and

intensity of nucleic acid staining in nuclei (Figure 2).

Compared to Brand B slides, which were tissue-culture-

treated, cells grown on Millicell EZ slides (untreated) exhibited

slightly less attachment.

Figure 2. NIH 3T3 cells cultured on Brand B 4-well glass chambered slide (A) and Millicell 4-well glass EZ slide (B). Actin (red) and nucleic acids (green) are visualized by immunocytochemistry.

A.

B.

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Similar analysis of actin polymers and nucleic acid

staining in CHO-K1 cells showed equivalent cell growth,

attachment, and morphology for Millicell EZ slides and Brand B

chambered slides (Figure 3).

ReNcell neural stem cells displayed no difference in cell

proliferation or morphology between Millicell EZ slides and

Brand B chambered slides (Figure 4A and 4D). These neural

stem cells appeared to maintain multipotency, as evidenced

by robust expression of nestin and Sox-2, which are neural

stem cell markers that are not expressed in differentiated

cells.

In conclusion, Millicell EZ slides improve experimental

throughput and save laboratory space without compromising

accuracy of data. Given that cells grow well and maintain

expected phenotypes on Millicell EZ slides, the greatly

increased convenience of this cell analysis format is a

compelling reason for performing immunocytochemistry

exclusively on Millicell EZ slides.

Figure 3. CHO-K1 cells cultured on Brand B 4-well glass chambered slide (A), Millicell 4-well glass EZ slide (B), Brand B 8-well glass chambered slide (C), and Millicell 4-well glass EZ slide (D). Actin (red) and nucleic acids (green) are visualized by immunocytochemistry.

Figure 4. ReNcell CX cells cultured on Millicell 8-well glass EZ slides (A, B, C) and Brand B 8-well glass chambered slides (D, E, F). Cells show staining for nestin (red, B and E) and Sox-2 (red, C and F). Nuclei (blue) are stained with DAPI.

A.

C.

B.

D.

A.

D.

B.

E.

C.

F.

Millipore, Millicell, and Advancing Life Science Together are registered trademarks of Millipore Corporation. Alexa Fluor and SYTO are registered trademarks of Life Technologies, Inc. ReNcell is a registered trademark of by ReNeuron Ltd.Lit. No. AN0056EN00 Printed in U.S.A. 05/10 BS-GEN-10-03204 © 2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

www.millipore.com

TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

In Europe, please call Customer Service:

France: 0825.045.645

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For other countries across Europe and the world,

please visit www.millipore.com/offices.

For Technical Service, please visit www.millipore.com/techservice.

Description Qty/Pk Catalogue No.

Millicell EZ slide (4-well glass) 16 PEZGS0416

96 PEZGS0496

Millicell EZ slide (8-well glass) 16 PEZGS0816

96 PEZGS0896

ORDERING INFORMATION

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