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®
HiPer Animal Tissue Culture Teaching Kit
Product Code: CCK005
Contents
Two vials of CHO cells
1. About the kit have been provided so that
2. Kit contents and storage instructions one vial can be used a back up vial in case of failure
3. Materials required but not provided in the kit to revive or propagate the cells in the other vial.
4. Aseptic techniques and good cell culture practices
5. Instructions for use CHO cells if cryopreserved at -180°C have an
6. Protocols indefinite shelf life. Shelf life of all other reagents is
6.1 Preparation of complete medium minimum one year from the date of manufacture.
6.2 Thawing of cryopreserved cells
6.3 Sub-culturing of the cells b. Leibovitz’s Medium: : Leibovitz’s Medium is a
6.4 Estimation of viability and enumeration of the cells
growth medium specifically designed to grow cells in
a CO2 free atmosphere. The standard sodium
1. About the kit bicarbonate/CO buffering system is replaced by a
2
combination of free base amino acids, phosphate
® buffers and higher levels of galactose and sodium
HiPer Animal Tissue Culture Teaching Kit has been
developed for teaching basic animal tissue culture pyruvate so that the medium does not require
techniques in educational organizations, where the supplementation with sodium bicarbonate and can be
sophisticated facilities required for culturing of animal used under conditions of free gaseous exchange with
cells may not be available. atmosphere.
CCK005, HiPer® c. Fetal Bovine Serum: Fetal Bovine Serum (FBS)
Animal Tissue Culture Teaching Kit is
sufficient to perform 10 - 12 subcultures post thaw. serves as a source of proteins, vitamins,
carbohydrates, lipids, hormones, growth factors,
Long term cryopreservation of cells without loss in minerals and trace elements. FBS contains growth
viability requires storage in liquid nitrogen atmosphere at factors which promote cell proliferation and has an
-180oC. Hence to use the kit most effectively, it is antitrypsin activity which helps in cell attachment.
recommended to thaw the cells and start the procedure
immediately after receipt. d. Antibiotic Antimycotic Solution: Antibiotic
Antimycotic solution helps to prevent microbial
In our experience, CHO cells lost viability when stored in contamination. This solution is a mixture of
the upper chamber (freezing compartment) of a frost-free Penicillin, Streptomycin and Amphotericin B in 0.9%
refrigerator. normal saline. It is effective against Gram positive
bacteria, Gram negative bacteria, Fungi and Yeast.
a. *Chinese Hamster Ovary (CHO) Cells: CHO cells
are fibroblast cells which are derived from the ovary e. Trypsin – EDTA Solution: Trypsin – EDTA
of the Chinese hamster. They grow as a monolayer Solution is a cell dissociation solution containing
and can be cultured in a CO independent atmosphere. Trypsin and EDTA in Dulbecco’s Phosphate Buffered
2
*Cells supplied with this kit are strictly for educational activity in teaching institutions and should not be used for any other
commercial purpose.
Saline. Trypsin is a serine protease commonly used g. Trypan Blue Solution: It is a vital stain. It selectively
for dissociation and disaggregation of adherent cells. stains the dead tissues or cells which after staining
Ethylenediaminetetraacetic acid (EDTA), a chelating appear blue in colour. Live cells or tissues with intact
agent is added to enhance enzymatic activity of cell membrane are selective for the compounds that
trypsin solution. EDTA acts by chelating calcium and pass through the membrane as a result of which trypan
magnesium ions that enhance cell to cell as well as blue is excluded by viable cells and they remain
cell to flask adhesion. colourless.
2.
f. Dulbecco’s Phosphate Buffered Saline (DPBS): h. Tissue Culture Flasks: Tissue Culture Flasks
DPBS is a balanced mixture of synthetic salts that provided in this kit have a vented cap, surface area of
maintains pH and osmotic pressure in the medium and 2
25cm and a working volume of 5ml. These are
provides adequate concentration of essential organic surface treated flasks which are specifically used to
ions to the cells. It is used to wash the monolayer of culture adherent cells.
cells as it is free of calcium and magnesium. It does not
hinder the trypsin activity.
7.
2. Kit contents and storage
8.
On receipt, remove the contents of the kit and place them in appropriate storage locations as per recommended
9.
storage temperature. 10.
Contents Quantity Storeat
12.
Code Description
13.
Cryopreserved Chinese Hamster Ovary 2 vials -20°C / -
CCK005(A) Cells 14. Each vial contains ∗
6 170°C
cells/ml
15. 2X10
Leibovitz's L-15 Medium o
CCK005(B) With L-Glutamine 16. 90ml 2 - 8 C
Fetal Bovine Serum 17. o
CCK005(C) 18. 10ml -20 C
Antibiotic Antimycotic Solution 100X
19.
w/10,000U Penicillin, 10mg Streptomycin
CCK005(D) and 25µg Amphotericin in 0.9% normal 1ml -20°C
saline 20.
Trypsin-EDTA Solution 1X 21.
•
CCK005(E) 0.25% Trypsin and 0.02% EDTA in -20°C
22. 15ml
Dulbecco's Phosphate Buffered Saline
23.
Dulbecco's Phosphate Buffered Saline 1X • 15-30°C
CCK005(F) Without calcium and magnesium 24. 25ml
Trypan Blue 0.5% solution in Dulbecco's
25. • 15-30°C
CCK005(G) Phosphate Buffered Saline 26. 10ml
Tissue culture flask 10Nos 15-30°C
CCK005(H) 2 27.
25cm , Surface treated, vented cap
28.
∗ 29.
For long term storage 30.
• .
Quantities supplied in excess to compensate operational losses
31.
3. Materials required but not provided in the kit c. Before starting tissue culture work switch on the UV
light in the cabinet for 15-20 minutes.
3.1 Equipments d. Keep all the work surfaces free from clutter.
• Laminar air flow hood e. All reagent and media bottles should be labeled
• Incubator at 37°C correctly with name and date of preparation and should
• Inverted microscope with 10X objective be kept at recommended storage temperatures.
• Hemocytometer with cover slip f. Clean the working area of the laminar air flow hood
• Centrifuge with 70% isopropanol.
• Water bath at 37°C g. Prior to starting work all reagent and media bottles,
pipettes, tip boxes should be sprayed with 70%
isopropanol.
3.2 Consumables h. Arrange the work station in such a way that you have
an easy access to all the items and a wide clear space in
• Micropipett the centre of the bench.
e
• Tips (1000µl, 200µl) and tip boxes i. Keep all the reagents and media bottles to the left hand
• Serological pipettes side of work station and the consumables and discard
• 15ml centrifuge tubes beaker to the right hand side of work station for
• 1ml eppendorf tubes efficient working.
• Pipette aid (LA692) j. While working do not contaminate the gloves by
• Disposable gloves touching anything outside the cabinet (especially face
• Lab coat and hair). In case they become contaminated then,
• Isopropanol spray respray with 70% isopropanol before proceeding.
• Tissue paper k. In case of any spillage while working, mop up
immediately and swab the area with 70% isopropanol.
4. Aseptic techniques and good cell culture l. Avoid rapid movement within and immediately outside
practices the cabinet. Slow movement will allow the air within
the cabinet to circulate properly.
a. Use Personal Protective Equipment (PPE), (laboratory m. Avoid speaking, sneezing and coughing while working
coat, gloves and eye protection) at all times while in the cabinet to prevent the contamination.
working in a cell culture lab. Use head caps to cover n. Pipette tips, waste reagents and waste medium should
hair. be discarded carefully into a separate discard beaker.
b. PPE for tissue culture facility should be kept separate o. Once the work is finished, clear the working area and
from PPE worn in general laboratory environment. clean with 70% isopropanol.
5. Instructions for use
Prepare complete medium as per protocol no. 6.1
Thaw the frozen cells as per protocol no. 6.2
Seed the cells in T-25 flask as
described in protocol no. 6.2, step (f)
After 2hrs
Check for cell attachment
Cells attached Cells not attached
Incubate the cells for another two hours
Give medium change as described in protocol no. 6.2, step (j)
o
Incubate at 37 C
Flask will be 80 – 90% confluent
Sub-culture the cells as per protocol no 6.3
Estimate the viability & enumerate the cells as per protocol no 6.4
Seed the cells in T-25 flask for further maintenance as per protocol no 6.3.2
Flask will be 80 – 90% confluent
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