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- : SF003
- : 1 kit
ESGRO Complete™ Derivation Kit
Description:
ESGRO Complete™ Derivation Kit
Trade Name:
- Esgro
- Chemicon (Millipore)
Qty/Pk:
1 kit
Product Overview:
ESGRO Complete™ Derivation Kit is intended for the derivation of mouse ES cells from delayed blastocysts in serum-free conditions.
Key Applications:
Stem Cell Culture
Application Notes:
Contains sufficient material and protocol for the treatment of a minimum of five mice, which is sufficient to generate delayed blastocysts to yield on average five ES cell lines. Designed for researchers experienced in animal work and micromanipulation of embryos.
All animal work must be conducted under the rules and regulations of the country in which you are operating. All procedures with primary cell lines should be conducted in a quarantine area until lines are tested for mycoplasma and other pathogens.
PROTOCOL:
1. Set up matings and monitor for plugged females the following morning. The most common strain of mice used is 129/Sv or 129/Ola, but ES cell lines can be derived from other strains such as C57BL/6.
2. Exactly 2 days after the plugs are detected inject the plugged females with 100 μL of Injection solution 1 as an IP injection, and 100 μL of Injection solution 2 subcutaneously. Make sure Solutions 1 and 2 are made up as described above. Failure do this could be lethal to the animals.
3. After 4 to 5 days flush the delayed embryos from the uterus with Basal Medium (part no. SF002-100) into a 6 cm2 tissue culture dish using standard techniques, then place the blastocysts into non-coated center well organ culture dish (do not add the gelatin to the plasticware at this stage). Using transfer needle A, transfer the embryos into a gelatin coated well dish containing DBIM (part no. SF010) pre-incubated in the tissue culture incubator. Embryos should then be incubated at 37°C in a 7% CO2 tissue culture incubator in this medium for a further 4-5 days (without media change). During this time the morphology of the embryo will change. The embryo may float freely or attach lightly. It is normal not to see any form of cell outgrowth at this stage in serum-free conditions.
4. Set up 4 well dishes in the flow hood by coating with Gelatin Solution (part no. SF008-100) for 30 minutes. Remove the buffer and allow to dry for 15 minutes. To these add Clonal Grade Medium (part no. SF001-100) and pre-incubate in the tissue culture incubator.
5. Place 50 μL drops of Trypsin solution (part no. SF007-20) onto a non-coated 6 well tissue culture dish. Remove each delayed blastocyst into these drops one at a time. Leave the embryo for 2-3 minutes only. Watch for morphology change by microscopy. Individual cell structures will become visible in the embryo and although some dissociation of cells will occur during this time, the embryo should remain largely intact. NOTE: Do not use standard trypsin.
6. Remove the embryo from the Trypsin solution (part no. SF007-20) with transfer needle B. Be careful to transfer the embryo in the smallest quantity of trypsin possible. To do this pre-fill the needle with a small amount of Clonal Grade Medium and then in the smallest possible volume of Trypsin to take up the embryo into the needle using the embryo handling pipette. Next place in the 4 well plates made previously and using the same needle vigorously pipette up and down until the embryo disassociates to almost a single cell suspension. In some cases the smaller bore needle C is required instead of needle B.
7. Incubate the 4 well dishes at 37°C in 7% CO2 for five or more days without media change until colonies are formed.
8. Prepare the 96 well plate by coating in Gelatin solution for 30 minutes, then removing the buffer and allowing to dry for 15 minutes. Add Clonal Grade Medium and pre-incubate in the tissue culture incubator.
9. Prepare an uncoated 6 cm2 tissue culture dish with drops of Trypsin Solution (part no. SF007-20) as done previously. From the 4 well dish remove the colonies by using transfer needle A and place them in the trypsin drops for 2-3 minutes only. Watch for morphology change by microscopy, again the colony will largely be intact. Now move the colony using needle B (pre-filled with Clonal Grade Medium and taking only the smallest possible amount of the trypsin solution - needle C may be required if the colonies are small). Expel the colonies into wells on the 96 well plate and disaggregate the colony by gently pipetting up and down. Incubate until the colonies are sufficiently large for further passage. Regularly monitor growth of the colonies by microscopy. NOTE: Do not use standard trypsin.
10. When ready to passage colonies prepare coated 4 well plates by coating in the Gelatin Solution for 15 minutes, removing the buffer and allowing to dry for 25 minutes. Remove the media from the cells and add 100 μL Trypsin Solution (part no. SF007-20) and incubate for 2 minutes in the tissue culture incubator. Disaggregate by pipetting gently using a Gilson or similar hand pipette. Remove contents of the well and add to a universal tube containing 5 mL of Basal Medium to neutralize the trypsin. Spin the universal tube at 1500 rpm in a bench top centrifuge. Carefully remove the supernatant and resuspend the cell pellet in a small volume of Clonal Grade Medium and transfer this to the prepared gelatin coated 4 well dish. NOTE: Do not use standard trypsin.
11. Continue to expand the colonies into larger coated wells and dishes. For instance, from 4 well plate to a 12 well plate to a small tissue culture flask and then a medium size flask.
12. When cell culture is established seed some cells for freezing into small coated tissue culture flask. Cells to be frozen should be in late log phase growth.
13. Thaw Complete Freezing Medium (part no. SF005) completely and mix well by gently swirling bottle. Keep freezing medium on ice during use.
14. Monolayers will need to be dissociated. After dissociation, cells are resuspended in Clonal Grade Medium and counted to determine viability and number.
15. Resuspend the cells in Cell Culture Freezing Medium at a concentration of ~4 x106 cells/mL. Freeze 1 mL of cells/vial. After the cells have been resuspended and aliquoted into appropriate cryogenic storage vials, they can be placed on dry ice and your normal freeze down procedure should be started within five minutes.
16. Cells must be stored at or below -80°C. For long term storage the cells should be stored in ultra-low temperature freezers (-150°C), or in liquid nitrogen (-196°C).
17. Thawing of cryopreserved cells should be as follows:
a. Thaw cells quickly in a 37°C water bath.
b. Dilute one vial of cells into 10 mL of Clonal Grade Medium.
c. Gently mix the cells in the growth media.
d. Gently pellet cells and remove the medium above the pellet.
e. Resuspend the cells in Clonal Grade Medium, dilute to the appropriate concentration, and plate into the appropriate vessel.
All animal work must be conducted under the rules and regulations of the country in which you are operating. All procedures with primary cell lines should be conducted in a quarantine area until lines are tested for mycoplasma and other pathogens.
PROTOCOL:
1. Set up matings and monitor for plugged females the following morning. The most common strain of mice used is 129/Sv or 129/Ola, but ES cell lines can be derived from other strains such as C57BL/6.
2. Exactly 2 days after the plugs are detected inject the plugged females with 100 μL of Injection solution 1 as an IP injection, and 100 μL of Injection solution 2 subcutaneously. Make sure Solutions 1 and 2 are made up as described above. Failure do this could be lethal to the animals.
3. After 4 to 5 days flush the delayed embryos from the uterus with Basal Medium (part no. SF002-100) into a 6 cm2 tissue culture dish using standard techniques, then place the blastocysts into non-coated center well organ culture dish (do not add the gelatin to the plasticware at this stage). Using transfer needle A, transfer the embryos into a gelatin coated well dish containing DBIM (part no. SF010) pre-incubated in the tissue culture incubator. Embryos should then be incubated at 37°C in a 7% CO2 tissue culture incubator in this medium for a further 4-5 days (without media change). During this time the morphology of the embryo will change. The embryo may float freely or attach lightly. It is normal not to see any form of cell outgrowth at this stage in serum-free conditions.
4. Set up 4 well dishes in the flow hood by coating with Gelatin Solution (part no. SF008-100) for 30 minutes. Remove the buffer and allow to dry for 15 minutes. To these add Clonal Grade Medium (part no. SF001-100) and pre-incubate in the tissue culture incubator.
5. Place 50 μL drops of Trypsin solution (part no. SF007-20) onto a non-coated 6 well tissue culture dish. Remove each delayed blastocyst into these drops one at a time. Leave the embryo for 2-3 minutes only. Watch for morphology change by microscopy. Individual cell structures will become visible in the embryo and although some dissociation of cells will occur during this time, the embryo should remain largely intact. NOTE: Do not use standard trypsin.
6. Remove the embryo from the Trypsin solution (part no. SF007-20) with transfer needle B. Be careful to transfer the embryo in the smallest quantity of trypsin possible. To do this pre-fill the needle with a small amount of Clonal Grade Medium and then in the smallest possible volume of Trypsin to take up the embryo into the needle using the embryo handling pipette. Next place in the 4 well plates made previously and using the same needle vigorously pipette up and down until the embryo disassociates to almost a single cell suspension. In some cases the smaller bore needle C is required instead of needle B.
7. Incubate the 4 well dishes at 37°C in 7% CO2 for five or more days without media change until colonies are formed.
8. Prepare the 96 well plate by coating in Gelatin solution for 30 minutes, then removing the buffer and allowing to dry for 15 minutes. Add Clonal Grade Medium and pre-incubate in the tissue culture incubator.
9. Prepare an uncoated 6 cm2 tissue culture dish with drops of Trypsin Solution (part no. SF007-20) as done previously. From the 4 well dish remove the colonies by using transfer needle A and place them in the trypsin drops for 2-3 minutes only. Watch for morphology change by microscopy, again the colony will largely be intact. Now move the colony using needle B (pre-filled with Clonal Grade Medium and taking only the smallest possible amount of the trypsin solution - needle C may be required if the colonies are small). Expel the colonies into wells on the 96 well plate and disaggregate the colony by gently pipetting up and down. Incubate until the colonies are sufficiently large for further passage. Regularly monitor growth of the colonies by microscopy. NOTE: Do not use standard trypsin.
10. When ready to passage colonies prepare coated 4 well plates by coating in the Gelatin Solution for 15 minutes, removing the buffer and allowing to dry for 25 minutes. Remove the media from the cells and add 100 μL Trypsin Solution (part no. SF007-20) and incubate for 2 minutes in the tissue culture incubator. Disaggregate by pipetting gently using a Gilson or similar hand pipette. Remove contents of the well and add to a universal tube containing 5 mL of Basal Medium to neutralize the trypsin. Spin the universal tube at 1500 rpm in a bench top centrifuge. Carefully remove the supernatant and resuspend the cell pellet in a small volume of Clonal Grade Medium and transfer this to the prepared gelatin coated 4 well dish. NOTE: Do not use standard trypsin.
11. Continue to expand the colonies into larger coated wells and dishes. For instance, from 4 well plate to a 12 well plate to a small tissue culture flask and then a medium size flask.
12. When cell culture is established seed some cells for freezing into small coated tissue culture flask. Cells to be frozen should be in late log phase growth.
13. Thaw Complete Freezing Medium (part no. SF005) completely and mix well by gently swirling bottle. Keep freezing medium on ice during use.
14. Monolayers will need to be dissociated. After dissociation, cells are resuspended in Clonal Grade Medium and counted to determine viability and number.
15. Resuspend the cells in Cell Culture Freezing Medium at a concentration of ~4 x106 cells/mL. Freeze 1 mL of cells/vial. After the cells have been resuspended and aliquoted into appropriate cryogenic storage vials, they can be placed on dry ice and your normal freeze down procedure should be started within five minutes.
16. Cells must be stored at or below -80°C. For long term storage the cells should be stored in ultra-low temperature freezers (-150°C), or in liquid nitrogen (-196°C).
17. Thawing of cryopreserved cells should be as follows:
a. Thaw cells quickly in a 37°C water bath.
b. Dilute one vial of cells into 10 mL of Clonal Grade Medium.
c. Gently mix the cells in the growth media.
d. Gently pellet cells and remove the medium above the pellet.
e. Resuspend the cells in Clonal Grade Medium, dilute to the appropriate concentration, and plate into the appropriate vessel.
Species Reactivity:
Mouse
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Stem Cell Type:
- Mouse Embryonic Stem Cells
- Induced Pluripotent Stem Cells
Stem Cell Workflow Stage:
- Cells/Isolation
- Maintenance & Expansion
Components:
- Clonal Grade Medium (Part No. SF001-100): One 100 mL bottle
- Basal Medium (Part No. SF002-100): One 100 mL bottle
- DBIM: Delayed Blastocyst Incubation Medium (Part No. SF010): One 100 mL bottle
- Trypsin Solution (Part No. SF007-20): One 20 mL bottle
- Gelatin Solution (Part No. SF008-100): One 100 mL bottle
- Complete Freezing Medium (Part No. SF005): One 50 mL bottle
Brand Family:
Chemicon
Storage Conditions:
Refer to individual component storage and handling conditions.
Cell Culture Reagent Type:
Cell Culture Media
Product Name:
ESGRO Complete™ Derivation Kit
Materials Required but Not Delivered:
1. Injection solution 1: Tamoxifen (Sigma catalog no. T5648). To prepare stock solution (10 mg/mL), dissolve 100 mg in 10 mL ethanol. Store at 4°C for up to one month. To prepare working solution, dilute 100 μL of stock solution in 9.9 mL propylene glycol (100 μg/mL). Store at 4°C for up to one month. For injections use 100 μL per animal i.p.
2. Injection solution 2: Depo-Provera (Medroxyprogesterone 17-acetate) (Sigma catalog no. M1629). Prepare a stock 150 mg/mL aqueous suspension using sterile distilled water. Dilute with sterile phosphate buffered saline to a concentration of 30 mg/mL. Store at 4°C for up to one year. For injections use 100 μL per animal subcutaneously.
3. These needles need to be created by using a micropipette puller:
Transfer needles A: For the handling of Blastocysts and outgrowths
Transfer needles B: Thin needles for disaggregating outgrowths and colonies
Transfer needles C: Extra thin for disaggregating outgrowths and colonies
4. Embryo handling pipette
5. 30 mL universal tube (Sterilin)
6. 4 well tissue culture plates
7. 6 well tissue culture plates
8. 12 well tissue culture plates
9. 96 well tissue culture plates
10. Center well organ culture dishes (Falcon 35-3037)
11. 6 cm2 tissue culture dish
12. 25 cm2 tissue culture flasks
13. Tissue culture flow hood
14. Tissue culture incubator humidified at 7% CO2 at 37°C
15. Sterile phosphate buffered saline
16. Syringes and small needles for injections
17. Stereomicroscope
18. Inverted microscope
2. Injection solution 2: Depo-Provera (Medroxyprogesterone 17-acetate) (Sigma catalog no. M1629). Prepare a stock 150 mg/mL aqueous suspension using sterile distilled water. Dilute with sterile phosphate buffered saline to a concentration of 30 mg/mL. Store at 4°C for up to one year. For injections use 100 μL per animal subcutaneously.
3. These needles need to be created by using a micropipette puller:
Transfer needles A: For the handling of Blastocysts and outgrowths
Transfer needles B: Thin needles for disaggregating outgrowths and colonies
Transfer needles C: Extra thin for disaggregating outgrowths and colonies
4. Embryo handling pipette
5. 30 mL universal tube (Sterilin)
6. 4 well tissue culture plates
7. 6 well tissue culture plates
8. 12 well tissue culture plates
9. 96 well tissue culture plates
10. Center well organ culture dishes (Falcon 35-3037)
11. 6 cm2 tissue culture dish
12. 25 cm2 tissue culture flasks
13. Tissue culture flow hood
14. Tissue culture incubator humidified at 7% CO2 at 37°C
15. Sterile phosphate buffered saline
16. Syringes and small needles for injections
17. Stereomicroscope
18. Inverted microscope