HBSB-5030                    200 Units per case.

STERILE R, by Gamma irradiation, 10 kGy minimum.
NOTE:  Products are sterile, until packaging is opened or damaged.

*  With the WillCo-dish®KIT dishes, you need the "ASSEMBLY DEVICE", Series DEV-5030, to bond the coverslip 
   "Safe, Easy, Accurately/precise and Quick". With the "kit", a 'Do-It-Yourself' WillCo-dish®, you also save a lot of 
    money! The Device comes separate.

If you have just any questions concerning our products or packaging, please do not hesitate to contact us.

Your own shipper

In case you take care of your own transport, your shipper may want
to have the following information, on your goods.

SIZES, VOLUMES & WEIGHTS:

GWST-Series & GWSB-Series cases (Blister-Pack)
1 Case:  Size: 36 x 28 x 19 cm. / Volume: 0.02 m3 / Weight:  1.95 kg.
2 Case:  Size: 36 x 28 x 39 cm. / Volume: 0.04 m3 / Weight:  3.90 kg.
3 Case:  Size: 36 x 28 x 59 cm. / Volume: 0.06 m3 / Weight:  5.90 kg.
4 Case:  Size: 36 x 28 x 66 cm. / Volume: 0.08 m3 / Weight:  7.90 kg.
5 Case:  Size: 36 x 56 x 59 cm. / Volume: 0.12 m3 / Weight:  8.85 kg. (We add one empty case, for the right 3+3 shape).

HBST-Series & HBSB-Series cases (Pouch-Pack)
1 Case:  Size: 26 x 22 x 18 cm. / Volume: 0.01 m3   / Weight:  1.60 kg.
2 Case:  Size: 26 x 22 x 36 cm. / Volume: 0.02 m3   / Weight:  3.30 kg.
3 Case:  Size: 26 x 22 x 54 cm. / Volume: 0.03 m3   / Weight:  4.90 kg.
4 Case:  Size: 43 x 35 x 25 cm. / Volume: 0.037 m3 / Weight:  6.70 kg.
5 Case:  Size: 53 x 43 x 25 cm. / Volume: 0.057 m3 / Weight:  8.95 kg. (We add one empty case, for the right 3+3 shape).

WillCo-dish®KIT-Series cases (KIT-Pack)
1 Case:  Size: 36 x 28 x 19 cm. / Volume: 0.02 m3 /  Weight:  2.45 kg.
2 Case:  Size: 36 x 28 x 39 cm. / Volume: 0.04 m3 /  Weight:  4.90 kg.
3 Case:  Size: 36 x 28 x 59 cm. / Volume: 0.06 m3 /  Weight:  5.90 kg.
4 Case:  Size: 36 x 28 x 65 cm. / Volume: 0.08 m3 /  Weight:  9.90 kg.

Your WillCo Wells B.V. 'Customer Service-Team'

HBSB-5030

Dear Colleague,

For this WillCo-dish® Glass Bottom dish, 'Series HBSB-5030', we use diameter 31.4 mm. glass, manufactured
by SCHOTT. Supplier of our glass, is Thermo Fisher (Former: Gerhard Menzel GmbH.).
For more specifications of the borosilicate D-263 M glass, you may want to visit their web site, to find all applicable
information on the 'glass bottom' of our WillCo-dish®'es.


Thicknesses we use:

Class #1.5H:  170 micron +/- 5 micron, with flatness  (Ra) 0.005 mm. (H: Specially selected glass).

Class #1.5   :   Everage of 170 micron (160 - 190 micron), with (Ra) 0.01 mm., as well as (Ra) 0.005mm.
NOTE          :   We do not use coverglass with these specifications, in our standard production, anymore.

and:

Class #1      :   Everage of 140 micron (130 - 160 micron).
                        Flatness (Ra) 0.01 mm., only.
NOTE          :   For these specificatios, we receive requests, only rarely (twice a year). Delivery times are long.

Your WillCo Wells B.V. 'Customer Service-Team'

HBSB-5030

Flatness:

The way the word 'flatness' is used, when discussing glass bottom dishes, there are two definitions of 'flatness'.

In the first place, there is the flatness of the glass coverslip bottom of the dish, when we talk about the glass coverslip being positioned in the dish as such, that it is absolutely horizontal on - and flush with, the warming stage.

Glass coverslip horizontal:
Positioned flat or horizontal means, that the light of the microscope passes the glass bottom orthogonally (very important) and that your cells stay in focus, when you move the stage, to another position (E.g. but not limited too: Time Laps Microscopy).

Glass coverslip 'flush', with the stage:
When the glass coverslip is flush with the stage, it means that there is no air-buffer, between the glass and the warming stage. The WillCo-dish® design, was - and is, the first dish to have this exceptional design. This feature too is very important, because it ensures an even distribution of the heat, from the warming stage to the media/liquid, inside the dish. All cells will be evenly heated, which is essential for the well being of your precious cells and the success of your work!

Note: If this is not the case, if there is an air-buffer between the warming stage and the bottom of the glass coverslip, an uneven distribution of the temperature to your cells, will shorten the life of your cells and dramatically influence the results of your work.

Coverslip roughness:
Secondly, when discussing 'flatness' of the glass coverslip, we mean the surface flatness (Ra) or roughness of the surface of the glass. We offer two specifications; surface flatness  Ra 0.005 mm. (standard) and Ra 0.01 mm., which surface is twice as rough.
We use specially selected glass only, thickness #1.5H, 170 micron glass +/- 5 micron, as well as this Ra 0.005 mm. flatness, for all our standard production.

Your WillCo Wells B.V. 'Customer Service-Team'

HBSB-5030

Dear Colleague,

You may need your WillCo-dish® Glass Bottom dishes to have a coating, for better adherence of your cells. We do
not administer coatings on the glass of our WillCo-dish®es, because it is not allowed to use liquids in the high standard
clean room, where we manufacture our WillCo-dish®es. Besides, we only rarely received an inquiry, for coated glass.

Over the past twenty years, by far the most of our well respected WillCo-dish®USERS, took care of their own coatings,
the reason why we offer you some coating procedures, below. We welcome your suggestions, to further support your
Colleagues.

Coating procedures for glass coverslips
1.: COLLAGEN,
2.: POLYLYSINE and POLYORNITHINE,
3.: FIBRONECTIN,
4.: LAMININ.

1.: COLLAGEN

Collagen may be used to coat glass coverslips for the growth of epithelial, endothelial and muscle cells, neurons, PC12
and CHO cell lines.
Type I collagen is most often used for coating substrates for cell culture, because it is easily obtainable from rat tails. For
short term cultures, collagen can be simply applied to glass coverslips and allowed to dry.
1. Dilute collagen solution 1:10 - 1:50 with 30% ethanol and spread over surface of sterile glass coverslip.
2. Air dry in a tissue culture hood.
3. Cells can be seeded directly on the collagen surface.
4. Collagen coating prepared in this way tends to detach from the glass in long-term cultures.

Collagen IV is the major constituent of basement membrane and is therefore a more physiological coating for the culture
of many cell types.
For long-term cultures, collagen I and IV can be applied to glass coverslips by first coating the glass with polylysine or
polyornithine. This provides a more stable collagen coating.
1. Prepare polylysine or polyornithine (MW of 30,000 - 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer
    (pH 8.3). Filter sterilize.
2. Add enough solution to pool over surface of sterile glass coverslip.
3. Incubate 2-24 hours at room temperature.
4. Aspirate solution and wash coverslips 3 times with water.
5. Pool collagen solution, 100 ug/ml in water over surface of coverslip.
6. Incubate 4 - 16 hours.
7. Rinse once with media and seed with cells.

Alternatively, for long-term cultures, double layered collagen coatings can provide a stable coating.
1. Spread a couple of drops of sterile collagen I solution on the sterile glass coverslip.
2. Immediately neutralize for 2 minutes with ammonium hydroxide vapors by placing the dish of coverslips
    in a covered dish containing filter paper wet with concentrated ammonium hydroxide. This will cause
    the collagen to gel.
3. Wash coverslips twice with sterile water.
4. Gently spread a couple of drops of collagen over the surface of the gelled collagen and air dry.
5. Use within a few hours for cell culture.

Gelatin can also be used for the culture of some cell types including glial cells.
1. Dissolve 100 mg gelatin in 100 ml water (triple glass distilled or RO).
2. Autoclave to sterilize.
3. While hot, thoroughly mix gelatin solution.
4. Add enough solution to pool over surface of sterile glass coverslip.
5. Chill for 2-24 hours at 4oC.
6. Remove gelatin by aspiration and add sterile water.
7. Dishes can be stored for up to one week at 4oC.
    Remove water immediately before use for cell culture

2.: POLYLYSINE AND POLYORNITHINE:
Nearly all types of cells adhere to these polymers of basic amino acids. They are particularly useful for the culture of CNS
neurons.
The L- or D-isomers can be used for cell attachment, however, the D-isomer may be preferred because it is not subject to
breakdown by proteases released by cells.
1. Prepare polylysine or polyornithine (MW of 30,000 - 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer  (pH 8.3). Filter
    sterilize.
2. Add enough solution to pool over surface of sterile glass coverslip.
3. Incubate 2-24 hours at room temperature.
4. Aspirate solution and wash coverslips 3 times with media or PBS.
5. Immediately add cell suspension or growth media.

3.: FIBRONECTIN:
Fibronectin is an extracellular matrix constituent use for the culture of endothelial cells, fibroblasts, neurons and CHO cells.
1. Stock solution can be prepared by dissolving 1 mg/ml fibronectin in PBS. Filter sterilize and freeze in aliquots.
2. Diluted stock solution to 50-100 ug/ml in basal medium or PBS.
3. Add enough solution to pool over surface of sterile glass coverslip.
4. Incubate for 30-45 min at room temperature.
5. Aspirate to remove fibronectin and rinse coverslips with media or PBS.
6. Immediately add cell suspension or growth media. Do not allow coating to dry.

4.: LAMININ:
Laminin is an extracellular matrix constituent used for the culture of neurons, epithelial cells, leukocytes, myoblasts and
CHO cells.
1. Stock solution can be prepared by dissolving 1 mg/ml laminin in PBS. Filter sterilize and freeze in aliquots.
2. Diluted stock solution to 10-100 ug/ml in basal medium or PBS.
3. Add enough solution to pool over surface of sterile glass coverslip.
4. Incubate several hours at room temperature.
5. Aspirate to remove laminin and rinse coverslips with media or PBS.
6. Immediately add cell suspension or growth media. Do not allow coating to dry.
7. Coating the glass coverslip first with polylysine or polyornithine and then laminin may increase the
    concentration of laminin applied using this method.