Human Skin Organoid Dissociation Reagent
Human Skin Organoid Dissociation Reagent
Price:
Regular price
$233 USD
Regular price
Sale price
$233 USD
Unit price
per
For shipping services or bulk orders, you may request a quotation.
Secure checkout with
View full details
Product Details
Product Details
Product Specification
| Usage |
Own reagents required: 1. horizontal rotor centrifuge ( Can be reduced to 4℃) 2. biological safety cabinet / ultra-clean workbench 3. CO2 incubator (5%CO2 ， 37℃) 4. Low temperature operating table 5. refrigerator ( 2-8°C ） 6. water bath / metal bath 7. pipette 8. Cytometer 9. inverted microscope Reagent dispensing: Tissue digestive juice preparation 1. Skin tissue digestive juice before use Ⅰ From -20 ℃ Taking it out of the environment, placing it at room temperature to melt it, turning the bottle upside down several times after it is completely dissolved to fully mix the liquid; 2.5 mL skin tissue digestive fluid Ⅰ Add basic medium ( DMEM ) Set the volume to 12.5 mL After configuration, record the preparation date on the label. 2- 8℃ Store in the refrigerator away from light; Note: Skin tissue digestive fluid Ⅰ After preparation, it is recommended to... 1 Used within a month; 3. Formulation contains 5%FBS 、 1% penicillin - streptomycin DMEM The neutralization solution was used to stop the digestion. Note: The centrifuge tubes, gun tips, etc. used in this experiment must be washed in advance with a special washing solution. Preparation before operation: 1. The centrifuge temperature is set to 4℃ Pre-cooling; 2. The sampling gun tip was placed in -20 ℃ Precool and remove before sample addition; 3.24 The orifice plate is placed in 37℃ Preheating the constant temperature incubator; 4. The matrix glue is placed on top of ice or 4℃ refrigerator 2-3 h melt; Note: Keep the matrix glue in place during use. 4℃ The following (it is recommended to store on ice) will cause the matrix glue to solidify and become unusable due to increased temperature. 5. The centrifuge tube is washed by the washing liquid and then placed on ice for precooling; 6. The dispensed human epidermal organoid medium was equilibrated to room temperature; Note: If the matrix gel needs to be diluted, the dilution medium can be placed in... 4℃ Pre-cooling; 7.DPBS Place on ice to precool. Operating instructions: Tissue Digestion: Attention! During the organoid procedure, all centrifuge tubes and pipette tips... Consumables that come into contact with organoids, such as disposable straws and cell screens, must be washed with a special washing solution before operation to reduce cell loss. 1. Flatten the annular foreskin tissue of the surgical sample on 10 cm In a petri dish, contain 1% penicillin - streptomycin DPBS Soaking in the solution, repeatedly rinsing to remove residual blood; 2. Remove subcutaneous structures of tissues, such as fat, blood vessels, etc., and rinse several times; 3. Cut the tissue evenly into 0.5-1.5 cm Square blocks of different sizes on the left and right sides, rinse again until the rinsing liquid is clear; 4. Place the washed small pieces of tissue in 10 mL Freshly prepared skin tissue digestive fluid Ⅰ In the middle, with the epidermis layer facing down, placed in 4℃ Digest overnight in the refrigerator; Note: The recommended digestion time is 12-14 h ； 5. After overnight digestion, the epidermis and dermis of the foreskin tissue were separated by gentle pulling with sterile forceps , collecting epidermal skin pieces; 6. Cut the epidermis into a paste with sterile surgical scissors and place it in a petri dish; 7. join 3 mL skin tissue digestive fluid Ⅱ , at 37℃ Digestion in the incubator 10 minutes; 8. Placing the culture dish under a microscope to observe the digestion; Note: You can gently tap the dish wall. A large number of floating and free cell bright spots can be seen under the microscope, which indicates that the digestion process is complete. Otherwise, the digestion time can be appropriately extended. 9. After digestion is complete, add 3 mL Contains 5%FBS 、 1% penicillin - streptomycin DMEM The neutralization solution terminates digestion; 10. Repeatedly and gently blowing to disperse the cell mass; 11. Moisturizing washing liquid 40 μm Cell screens and cell collection 50 mL a centrifuge tube; 12.40 μm Cell sieve filtration, and the filtrate is collected until washed. 50 mL in a centrifuge tube; 13.4℃ ， 400 g Centrifugation 5 min ； 14. Use DPBS resuspending the cell pellet; 15.4℃ ， 400 g Centrifugation 5 min 。 Organoid Culture: Attention! During the organoid procedure, all centrifuge tubes and pipette tips... Consumables such as disposable straws that come into contact with organoids must be washed with a special washing solution before operation to reduce cell loss. 1. Add an appropriate amount of pre-cooled matrix gum to the precipitate and mix gently. 10-15 Next; 2. Take an appropriate volume of the mixed solution and observe the density under a microscope; 3. Add an appropriate amount of matrix glue to adjust the density, and blow and mix well. 10-15 Next; Note: This step should be done as quickly as possible to avoid solidification of the matrix glue due to increased temperature; Do not generate air bubbles when blowing and beating; Please adjust the density appropriately according to the cell size; 4.50 μL / The matrix glue and organoid suspension were added dropwise to the hole. 24 The center of the orifice plate is hemispherical; 5.37℃ 、 5% CO2 Incubation in incubator 20 min Avoid shaking the culture plate during the process; 6. Add per well after incubation 500 μL human epidermal organoid medium; 7.37℃ 、 5% CO2 Continue cultivating in the incubator, each 2-3 Change the fresh medium once a day. Note: Observe daily after inoculation, and the entire operation should comply with aseptic operating procedures. |
||||||
| Description |
This kit is suitable for effective dissociation of primary keratinocytes, thereby rapidly obtaining cell masses or single-cell suspensions, and the obtained cells can be used for subsequent organoid culture.
|
||||||
| General Notes | 1.This product contains active agents. Please store the reagents strictly according to the kit instructions. Repeated freezing and thawing or improper storage will affect the experimental results. |
||||||
| Storage Temp. | Store at 2-8℃ in a dark place. Shelf life is 3 months. |