Marissa's Blog

Resuscitation of Frozen Cell Lines

noreply@blogspirit.com (Marissa) — Fri, 13 Jun 2008 10:16:15 +0200

Many cultures obtained from a culture collection, such as ECACC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic above 4oC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

A schematic diagram of "Resuscitation of Frozen Cell Lines"

Materials

Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and size of flask to resuscitation into.)

70% ethanol in water

DMSO

Equipment

Personal protective equipment (sterile gloves, Laboratory coat, safety visor)

Waterbath set to appropriate temperature

Glass Bottom Dishes

Microbiological safety cabinet at appropriate containment level

CO2 incubator

Pre labeled flasks

Marker Pen

Pipettes

ELISA plates

Ampule Rack

Tissue

Procedure

Read Technical data sheet to establish specific requirements for your cell line.

Prepare the flasks as appropriate (information on technical data sheet). Label with cell line name, passage number and date.

Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.

Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37oC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet.

Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.

Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (cell culture flasks prepared in Step 2).

Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere.

Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Key Points

Most text books recommend washing the thawed cells in media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks. See Protocol 7 for further details.

Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.

If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.25m filter.

For some cultures it is necessary to subculture before confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 (Prod. No. 93061524) cells is lost if they are allowed to reach confluence repeatedly.

Scouce: ECACC Handbook Protocol 2

Biousing Cell Culture Dishes List

noreply@blogspirit.com (Marissa) — Wed, 11 Jun 2008 09:07:04 +0200

Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a routine laboratory technique in the 1950s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.

--From wikipedia

Our products(such as cell culture dishes) are mostly for cell culture applications, review the category list below and click on a selection for product lists:

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-- 60mm Cell Culture Dish

-- 100mm Cell Culture Dish

-- 35mm Glass Bottom Culture Dish

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We emulate the management of pharmaceutical company to supervise the whole production process. A QA/QC system with SOPs and standard documentation management guarantees the quality of biousing products to the international standard.

Introduction of 96 Well ELISA Plate

noreply@blogspirit.com (Marissa) — Fri, 6 Jun 2008 10:52:46 +0200

p>A Microtiter plate (spelt Microtitre in Europe) or microplate is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories. A very common usage is in the enzyme-linked immunosorbent assay (ELISA), the basis of most modern medical diagnostic testing in humans and animals. A microplate typically has 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. -- from Wikipedia

Biousing Biotechnology Co., Ltd. is a professional biology hi-tech company that is focused on research, manufacturing and sales of high-level laboratory consumables, devices and reagents. Their featured ELISA produt is 96 well ELISA plate.

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Produced conform to the SBS-3d-standard exhibit finest workmanship. The binding capacity is 400-500ng/ cm2, CV<4%.

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The absolutely flat floor area, free from inclusions, guarantees the highest level of transparency. The detection background is lower than that of the other international brands.

The smoothness of Biousing cell culture plates is much better than the best-selling brands. This will greatly eliminate the system error when 96-well plates are used in ELISA.