FAQ’s: Glass Bottom Cultureware and 3D Tissue Models

A general procedure for their use follows.

1. Maintain sterility: Open dishes in a sterile environment (e.g. laminar flow hood).
2. Pre-equilibrate dishes: Incubate the dishes with culture medium. Pipet 2-3 ml of medium into the 35 mm dishes or 3-4 ml into the 50 mm dishes and incubate at 37° C for 15 minutes.
3. Add cell suspension to microwell: Remove the culture medium by aspiration and plate cells onto the glass surface. Pipet 250 µl of the cell suspension (cells suspended in culture medium) into the 10 mm diameter microwells, 500 µl of cell suspension into the 14-mm microwells, or 1 ml of cell suspension into the 20-mm wells. Incubate the dishes for 1 hour at 37° C.
4. Add additional medium: After 1 hour, gently fill the remainder of the dish with medium. Add 2-3 ml to the 35 mm dishes or 3-4 ml for the 50 mm dishes.

Note: After the initial one hour period to allow cells to attach to the glass surface, it is important to fill the dish to normal levels in order to minimize the effects of evaporation and to avoid inducing changes in osmolarity.

Note: For almost all microscopy applications, the No. 1.5 coverslip thickness is preferred.

1. Under sterile conditions, pipette 250 µl of 1 N HCl onto non-coated 10-mm glass bottom dishes (e.g. Part #’s: P35G-x-10-C or P50G-x-10-F); use 500 µl or 1 ml of 1 N HCl for the 14-mm and 20-mm glass bottom culture dish, respectively (e.g. Part #’s: P35G-x-14-C or P35G-x-20-C); .
2. After 15 minutes, decant the HCl and rinse the dish 3x with phosphate buffered saline (PBS) and 2x with ultra-pure H2O.
3.  Apply the coating to the dishes.
4. Add a similar volume of the medium in which you will plate your cells to pre-equilibrate the glass surface. Incubate the medium in glass bottom dishes for 15 min at 37°C. Remove the medium and then plate your cells.

To try a sample of non-coated glass bottom dishes, go to our free sample page.

If necessary (e.g. for long-term storage purposes), the coverslip can be removed using the following procedure:

1. Order Part # PDCF OS 30 (Fluid for removal of coverslips from glass bottom dishes)
2. Invert the cover of the dish.
3. Pipette 1.0 ml of fluid into the inverted cover.
4. Place the bottom of the dish onto the cover. Make sure that the liquid in the inverted cover is touching the bottom of the coverslip.
5. Allow the dish to sit in the fluid for 45 minutes at room temperature.
6. Dry the bottom of the coverslip with an absorbent paper towel.
7. Place the dish on a clean surface. Using forceps, press down on the inner edge of the coverslip to separate the coverslip from the dish.

Note: If the above procedure is followed, the PDCF OS 30 fluid will not contact the cells and will not disrupt cells on the coverslip or the staining thereof. Coverslips can be removed without breakage.

In order to approximate physiological conditions, the temperature of the medium contained within the glass bottom dishes can be controlled by using a microscope stage heater and an appropriate stage adapter.

For use with the P35G dishes (Corning 35 mm dishes): Culture dish heaters (part#: DH-35), microscope stage adapters (part#: SA-microscope type), heater controller (part#: TC324-B), and connector cable (part#: CC-28) are available from Warner Instrument Corporation. Information is available online at: http://www.warneronline.com/

The gridded coverslips allow one to refer to specific cells and follow them over time. For instance, individual cells can be microinjected, returned to the incubator, and observed at multiple time points since each cell can be identified with a unique alpha-numeric coordinate in the dish. Glass bottom dishes containing gridded Bellco Glass coverslips are available. Standard gridded dishes are designated as Part #’s: P35G-1.5-14-CGRD and P50G-1.5-14-FGRD.

Grid size: The grid on Part #’s P35G-1.5-14-CGRD and P50G-1.5-14-FGRD consists of 520 unique alphanumeric squares. Each square measures 600 microns x 600 microns. The line thickness is 20 microns.

Visualization of the grid: The grid should be readily observable using a 10X brightfield objective. After you locate the cell of interest, you can switch to a higher magnification or fluorescence objective. However, the grid will not be observable using higher power or fluorescence objectives.

I can’t see the grid: A confluent monolayer of cells will typically mask the grid making it difficult or impossible to visualize. If your cells are confluent, you can utilize Part # P35G-1.5-14-CGRD-D which places the grid on the outside of the dish where it is unaffected by the cells growing on the coverslip. To use this dish, find a cell of interest and then focus down to the bottom side of the coverslip to get its coordinates.

Correlative Light and Electron Microscopy (CLEM): The P35G-1.5-14-CGRD glass bottom dishes work nicely with CLEM; however, the P35G-1.5-14-CGRD-D is not recommended for CLEM because the cells and the grid are in a different focal plane.

1. Incident ultraviolet rays with wavelengths longer than 320 nm do not cause fluorescence.
2. Mercury lines at 334 and 365 nm do not create auto-fluorescence. (Note: For mercury illumination, filter out the mercury lines with wavelengths shorter than 313 nm to obtain best possible results.)
3. Refractive index (@ 20°C): nd= 1.5230 tolerance ± 0.0015
4. Abbe number V = 55.

For super-high resolution microscopy techniques, we offer high tolerance No. 1.5 coverslips designated in part #s as -0.170 (e.g. Glass bottom dish Part #: P35G-0.170-14-C).

The actual thickness of the glass coverslips depends on the Coverslip No./Part #, as follows:

伟德体育app下载 uses the highest quality, borosilicate German glass coverslips in its glass bottom dishes. The coverslip properties are as follows:

1. Highest hydrolytic resistance (hydrolytic class 1).
2. Excellent resistance to chemicals.
3. Emission of alkali approximately 15 to 24 µg Na2O/g glass.
4. Excellent properties for fluorescent microscopy.
5. Incident ultraviolet rays with wavelengths longer than 320 nm do not cause fluorescence.
6. Mercury lines at 334 and 365 nm do not create auto-fluorescence.(Note: For mercury illumination, filter out the mercury lines with wavelengths shorter than 313 nm
   to obtain best possible results.)
7. Refractive index (@ 20°C): nD ** = 1.5230 tolerance ± 0.0015.
8. Abbe number V = 55.

The depth of the micro-wells in the glass bottom dishes depends on the type of dish and the thickness of the dish bottom as follows:

1. Corning 35 mm: 0.70-0.75 mm
2. Falcon 50 mm: 1.00-1.10 mm
3. Falcon 60 mm: 1.15-1.20 mm
4. Falcon 100 mm: 0.90-1.00 mm*
5. Falcon 6-well plate: 1.45-1.55 mm
6. Falcon 12-well plate: 1.45-1.55 mm
7. Falcon 24-well plate: 1.10-1.20 mm
8. Falcon 96-well plate: 1.05-1.25 mm

The body of the glass bottom dishes and multi-well plates is made from polystyrene. Therefore, they have limited compatibility with organic solvents. Please see the chemical compatibility table.

The P35G-0.170-14-C dishes will improve picture quality (see Figure below) versus the P35G-1.5-14-C dishes for any high numerical aperture objective used in confocal, fluorescence, GSDIM, dSTORM, PALM total internal reflection (TIRF), and other high numerical aperture objectives. For example, quantitative measurements using P35G-0.170-14-C dishes gave z-resolution of +/- 9.5% while z-resolution in the P35G-1.5-14-C dishes gave z-resolution of +/- 17.3% (n=5).

A
B

Figure: Improved Z-axis resolution – Effect of high tolerance glass coverslips (in P35G-0.170-14-C glass bottom dishes) on imaging of sub-resolution beads using:
A) P35G-0.170-14-C and B) P35G-1.5-14-C glass bottom dishes.
5 µl of FITC labeled 175nm PS-Speck sub-resolution beads were added to well of the glass bottom dishes and allowed to dry. After drying, 200 ul of water were added and the beads were imaged using a Zeiss LSM510 confocal microscope equipped with an Olympus UPLSAPO 60x (NA=1.2) water immersion objective.

Figures and measurements courtesy of Teemu Ihalainen, Ph. D., University of Jyvaskyla, Finland (2008).

伟德体育app下载 produces a variety of normal (non-transformed), human cell-derived, 3-dimensional, organotypic in vitro tissue equivalents. This Web site, 伟德体育app下载.com, is devoted to providing detailed information about our in vitro human tissue equivalents. They are mitotically and metabolically active, closely mimic their in vivo counterparts, both structurally and biochemically, and do so with guaranteed reproducibility.

伟德体育app下载 also produces a line of Glass Bottom Culture Dishes. These dishes are used in confocal, polarized light, and fluorescence microscopy techniques, and are ideal for live cell microscopy applications.

• EpiDerm, our human epidermal tissue equivalent, consists of normal, human cell-derived epidermal keratinocytes that have been cultured to form a multilayered, highly differentiated model of the human epidermis. EpiDerm, also known generically as Reconstructed Human EpiDermis (RhE), has been in continuous production for over 15 years. There is also an “under-developed” version, EpiDerm-201
• EpiDermFT (EpiDerm Full Thickness), a dermal / epidermal human skin equivalent with a well-defined, fully functional basement membrane.
• MelanoDerm is a human skin equivalent composed of keratinocytes and melanocytes that have been cultured to form a multilayered, highly differentiated model of the human epidermis.
• Melanoma is a full thickness melanoma skin model composed of human malignant melanoma cells, normal, human-derived epidermal keratinocytes and normal, human-derived dermal fibroblasts that have been cultured to form a multilayered, highly differentiated epidermis with melanoma cells at various stages of CM malignancy.
• EpiOcular, our corneal model, consists of normal, human cell-derived epidermal keratinocytes that have been cultured to form a stratified, squamous epithelium similar to that found in the cornea.
• EpiAirway consists of normal, human cell-derived tracheal/bronchial epithelial cells that have been cultured to form a pseudo-stratified, highly differentiated model that closely resembles the epithelial tissue of the respiratory tract.
• EpiVaginal, derived from human ectocervico-vaginal (ECV) epithelial cells.
• EpiOral, our human buccal (inner cheek) equivalent,
• EpiGingival, our human gingival (gum) tissue equivalent.

• 9 mm diameter individual tissues, each tissue produced in a single well tissue culture plate insert (shipped in either 12 or 24 tissue kits with media)
• 22 mm diameter individual tissues, each tissue produced in a single well tissue culture plate insert (shipped in 6 tissue kits with media)

Tissues are also available in 24-well and 96-well high thru-put plates. Special configurations are also available for specific tissue types (ex. EpiAirway tissues produced in “snapwell” culture inserts for use in Using Diffusion Chambers).

There are many applications for each model. For example, our EpiDerm in vitro skin equivalent is used to determine and/or study the following: dermal corrosion, skin irritation (cutaneous toxicity), dermal phototoxicity, percutaneous absorption (drug permeability, transdermal drug delivery), inflammation, gene analysis, antioxidants, metabolism, apoptosis, antimicrobial peptides, and angiogenesis.

A very useful method to determine if 伟德体育app下载 In Vitro Products have been used in applications similar to your application is to search our list of Technical References.

Pharmacology/Toxicology pre-clinical applications for our in vitro human tissue equivalents include transdermal, transbuccal, transmucosal drug delivery; biocompatibility, toxicity studies; HIV, microbicide research; bioequivalence studies; lead optimization, etc.

An example of a pharm/tox application of 伟德体育app下载’s in vitro human tissue equivalents is as follows: after a new drug library has been processed thru a biochemical-based (enzyme) primary screening and a cell-based secondary screening, but PRIOR to performing pre-clinical animal studies, the remaining drug candidates are passed through a tertiary in vitro human tissue-based screening to determine optimum permeability and/or minimal toxicity characteristics. Those drug candidates that pass this tertiary screening then move onto the animal-based study, but that study will now require fewer animals, and can therefore be structured as a small confirmatory study to meet FDA (or other regulatory agency) requirements. Also, by performing the human tissue-based tertiary study prior to commencement of human clinical studies, the potential for cross-species extrapolation errors based on animal study results has also been significantly reduced.

Prior to purchasing our in vitro human tissue equivalents, you can purchase purified total RNA (control and treated) and/or Protein Lysate (control) from each of our human tissue equivalents to confirm the expression level of specific gene(s) or the presence/absence of specific protein(s).

For this reason, 伟德体育app下载 has spent many years developing the most reproducible in vitro human tissue equivalents available. For example, 伟德体育app下载 has over 15 YEARS of reproducibility data for the EpiDerm human skin equivalent.

Reproducibility is so important to the successful use of in vitro human tissue equivalents that 伟德体育app下载 GUARANTEES the reproducibility of ALL of its in vitro tissues.

There is a searchable database of TR citations/summaries on the 伟德体育app下载 Web site. There are over 700 TR’s (and growing!) listed in the database. Click here to go to the 伟德体育app下载 TR page.

From the ICCVAM Web site: “The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was established in 1997 by the Director of the National Institute of Environmental Health Sciences (NIEHS) to implement NIEHS directives in Public Law (P.L.) 103-43. This law directed NIEHS to develop and validate new test methods, and to establish criteria and processes for the validation and regulatory acceptance of toxicological testing methods. P.L. 106-545, the ICCVAM Authorization Act of 2000, established ICCVAM as a permanent committee. The Committee is composed of representatives from 15 Federal regulatory and research agencies; these agencies generate, use, or provide information from toxicity test methods for risk assessment purposes. The Committee coordinates cross-agency issues relating to development, validation, acceptance, and national/international harmonization of toxicological test methods.”

From the ECVAM Web site: “ECVAM (European Centre for the Validation of Alternative Methods) was created by a Communication from the Commission to the Council and the Parliament in October 1991, pointing to a requirement in Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, which requires that the Commission and the Member States should actively support the development, validation and acceptance of methods which could reduce, refine or replace the use of laboratory animals. ECVAM was established in 1992 as a unit of the Environment Institute, part of the Joint Research Centre, and was transferred, at that time, to the newly formed Institute for Health and Consumer Protection in Ispra, Italy in 1998.”

The entire validation process for an alternative method can take 8-10 years. The scientific portion of the process (formal pre-validation and validation studies) usually takes 4-6 years.

伟德体育app下载 has TWO alternative methods formally validated – EpiDerm for skin irritation testing and for dermal corrosion testing. Several others have completed major portions of the process – EpiOcular for ocular irritation (an alternative to the Draize Rabbit Eye Test) that is being sponsored by Colgate-Palmolive, and EpiDerm for percutaneous absorption testing.

伟德体育app下载 recently added Glass Bottom Multi-Well Plates to its glass bottom dish product offering.

Our dishes are known by many different names including glass bottom dishes, glass bottom culture dishes, glass bottom petri dishes, glass bottom microwell dishes, glass bottom sterile culture dishes, imaging dishes and coverslip bottom dishes. Regardless of what they are called, our 35 mm and 50 mm Glass Bottom Culture Dishes, and now our Glass Bottom Multi-well Plates, have become the de facto standard for high resolution microscopic imaging of in vitro cell cultures.

伟德体育app下载 Glass Bottom Dish and Muti-well Plate part numbers include: P35G-0-10-C, P35GC-0-10-C, P35G-0-14-C, P35GC-0-14-C, P35G-0-20-C, P35GC-1.0-14-C, P35G-1.0-14-C, P35GC-1.5-10-C, P35G-1.5-10-C, P35GC-1.5-14-C, P35G-1.5-14-C, P35GCOL-0-14-C, P35G-1.5-20-C, P35GCOL-1.0-14-C, P35G-1.5-14-CGRD, P35GCOL-1.5-14-C, P50G-0-14-F, P50GC-0-14-F, P50G-1.5-14-F, P50GC-1.5-14-F, P50G-2-14-FGRD, P06G-0-20-F, P12G-0-14-F, P06G-1.0-20-F, P12G-1.0-14-F, P06G-1.5-20-F, P12G-1.5-14-F, P24G-0-13-F, P96G-1.5-5-F, P24G-1.0-13-F, P24G-1.5-13-F, P96GC-1.5-5-F.

Glass Bottom Dishes with gridded glass coverslips are also available. Part numbers include P35G-1.5-14-CGRD, P50G-1.5-14-FGRD.

伟德体育app下载 Cell Culture Coverslip Kits

伟德体育app下载 Cell Culture Coverslip Kits come ready to use – coverslips placed in 35 mm petri dishes and pre-sterilized. Standard glass coverslip kits are available in the CSGK/F, CSGK/C and CSGK/N Coverslip Kit configurations. These kits, the CSGK/F in particular, are widely used in amniocentesis testing.