-
Categories
-
Pharmaceutical Intermediates
-
Active Pharmaceutical Ingredients
-
Food Additives
- Industrial Coatings
- Agrochemicals
- Dyes and Pigments
- Surfactant
- Flavors and Fragrances
- Chemical Reagents
- Catalyst and Auxiliary
- Natural Products
- Inorganic Chemistry
-
Organic Chemistry
-
Biochemical Engineering
- Analytical Chemistry
- Cosmetic Ingredient
-
Pharmaceutical Intermediates
Promotion
ECHEMI Mall
Wholesale
Weekly Price
Exhibition
News
-
Trade Service
cell surface moleculesantigen is one of the most commonly used methods of cell analysis. eBioscience provides near-complete fluorescence markers " and purified antibodies to mice, humans, and rat CD molecules and other membrane molecules, making it the perfect solution for experimenters' research.
1, experimental materials
|
one anti (tag or purification)
- Anti-CD16// 32 Antibodies are used to close the non-specific binding interference caused by Fc fragments (optional)
- fluorescent secondary (indirectly labeled dyeing)
- buffer: eBioscience FC Staining Buffer (Cat.No.00-4222) or PBS (PH7.4)
equipment: < a href" > > , < a href "> centrifuge ice box or refrigerator,streaming cell meter
.
II, Note
1. Antibodies are quickly centrifuged for a few seconds before use, so that all liquids are concentrated at the bottom of the tube.
2. Try to avoid aggregation between direct-labeled antibodies.
3. Antibodies should be stored in a light-avoiding 4 degrees C to avoid freezing.
4. The fixed preservation or long placement of the sample may affect the binding between the cell surface molecules and antibodies, so it is recommended to use fresh samples.
.3, operation method
< text-align: left;">A . Mouse lymphatic < a href > tissue cell surface antigen flow detection procedure
(i) sample preparation
1. Remove the mouse lymphatic tissue block, quickly soak in 10 ml of Staining Buffer, and grind with the piston of the syringe or use two pre-cooled slides to study into a single cell.
2. Transfer the suspension to a 50 ml conical tube and sit for a moment, sinking cell blocks and fragments to the bottom of the tube and passing through nylon nets such as Falcon Cat. No. 2350) < a href"" > filterinto a single-cell suspension.
300-400g centrifugation at 3.4 degrees C for 4-5 minutes, discarding the liquid.
4. If the sample is a spleen cell, add a certain amount of RBC lysis to lysate the red blood cells, or isolate the lymphocytes with a separator. If other samples are available, omit this procedure.
5. Add 50 ml of staining buffer to re-suspend the cell count and test for cell activity with Trypan Blue as needed.
6. centrifugal cell fluid and discarded for semen; 2× 107 /ml of staining buffer resurrevered cells. If a directly labeled resistance is used, incubate on ice with CD16/32 antibodies (0.5-1ug/106 cells) for 5-10 minutes.
(ii) cell fluorescence staining step
1. Add 50ul diluted resistance (antibodies diluted to a suitable concentration with Staining Buffer) to each flow test tube or plate microhole; and add 50ul Staining Buffer to the blank tube/hole or hose control tube/hole. For optimal antibody concentration testing, the recommended range is 2-0.03ug/106 cells.
< p style"text-align:left;">2. Add 50ul cell suspension (about 106 cells) to each tube/hole and mix gently.
3. Take a light-absorbing ice bath or incubate in a refrigerator at 4 degrees C for 20 minutes.
(Note: Some antibodies may take longer incubation time, suggested for pre-test groping by the experimenter)
4. After incubation, add Buffering Staining (2 ml per flow test tube and 200ul per micro-hole)
300-400g centrifugation at 5.4 degrees C for 5 minutes, and discard to the upper clear.
6. Repeat the washing process 3 times.
a) If one of the antibodies used is a fluorescent directly labeled antibody, it is tested after resusping the cells with 500ul Staining Buffer.
b) If one of the antibodies used is purified or biotin-labeled antibodies, add 50-100ul diluted fluorescent-labeled di-anti-or fluorescent-labeled affines (diluted to a suitable concentration with Staining Buffer) to each tube/hole and incubate for 15-30 minutes in an ice bath or in a 4c refrigerator. Wash the cells twice (refer to steps 4 and 5). The cells are then resealed and tested on the machine by 500ul Staining Buffer.
8.Analysis of test results.
(Note: To multicolor multiple cell surface antigens, incubate and wash the cells at the same time with the addition of fluorescently labeled antibodies; )
B . Flow detection steps for antigens on the surface of human extrinsic blood cells
1. Add 50ul fluorescent markers or diluted antibodies with Staining Buffer to a suitable concentration in each flow test tube or plate micro-hole; add 50ul Staining Buffer to blank tubes / holes or homolytic control tubes / holes. (eBioscience tests specification antibodies are ready-to-use antibodies, each test tube to add 20ul antibodies of this class)
2. Each tube is added 100ul whole blood, and gently mixed.
(Note: Some antibodies with lower binding capacity may require longer incubation times, it is recommended that the experimenter conduct pre-test groping)
4. Add 2 ml RBC lysis Buffer (previously preheat to room temperature) to mix well.
7. Wash cells once with 2 ml Of Staining Buffer.
a) If one of the antibodies used is fluorescent directly labeled antibodies, use 500ul S.
