Today we will talk about hybridoma cell culture~
·Hybridoma cell culture guide·
Operation steps and FAQs
In the field of molecular and cell biology, the culture of hybridoma cells is an important experimental technique. Hybridoma cells are formed by the fusion of B lymphocytes and myeloma cells, and can continuously secrete monoclonal antibodies, which is of great significance to basic research and clinical applications. This article will introduce the culture instructions, specific steps and solutions to common problems of hybridoma cells in detail.
Hybridoma cell culture instructions
Hybridoma cell culture aims to provide a suitable environment so that cells can grow stably and secrete monoclonal antibodies. During the culture process, temperature, humidity, culture medium components and gas environment need to be strictly controlled.
Culture steps
Preparation
1. Select appropriate hybridoma cell lines.
2. Prepare sterile culture medium and culture equipment.
3. Ensure the cleanliness and sterile operating conditions of the cell culture room.
Cell thawing and recovery
1. Take out the frozen hybridoma cells from liquid nitrogen.
2. Thaw cells quickly in a 37℃ water bath for about 1-2 minutes until the ice melts.
3. Slowly dilute the cell suspension with 10 times the volume of preheated culture medium and centrifuge at 300g for 5 minutes.
4. Discard the supernatant, resuspend the cells with fresh culture medium, and transfer to a culture flask.
Cell culture and passaging
1. Culture the cells in a 37℃, 5% CO2 incubator.
2. Check cell growth regularly, and generally change the medium every 2-3 days.
3. When the cells reach an appropriate density, subculture them, and the common subculture ratio is 1:3 to 1:5.
Monoclonal antibody production and detection
1. Select hybridoma cell lines with higher antibody secretion.
2. After the cells are cultured to a high density, collect the supernatant.
3. Detect the antibody concentration and activity by ELISA or Western Blot.
Related Products
【Genom Biology】RPMI-1640 series is widely used in the culture of mammals, special hematopoietic cells, normal or malignant proliferation of leukocytes, and hybridoma cells. Mainly used for suspension cell culture. Others such as K-562, HI-60, Jurkat, Daudi, IM-9, lymphoblasts, cell lymphoma cells, and HCT-15 epithelial cells can be used for reference.
Common Problems and Solutions
Low vitality after cell recovery
1. The recovery process should be as fast as possible to avoid long-term exposure of cells to a 37°C water bath.
2. Use fresh culture medium and sterile operation.
Slow cell growth
1. Check the composition and quality of the culture medium to ensure its suitability.
2. Check whether the CO2 concentration and culture temperature are stable in the appropriate range.
3. Change the culture medium regularly to avoid the accumulation of metabolic waste.
Cell contamination
1. Strictly perform aseptic operation and clean the incubator and workbench regularly.
2. Use antibiotics (such as penicillin and streptomycin) to prevent bacterial contamination.
3. If contamination is found, immediately discard the contaminated cells and culture medium, and thoroughly clean the culture equipment.
Low antibody yield
1. Select cell lines with high antibody production for culture.
2. Optimize culture conditions, such as culture medium formula and cell density.
3. Use cell lines with good stability to avoid gene drift caused by frequent subculture.
Hybridoma cell culture has important applications in monoclonal antibody production. Through standardized operating procedures and effective problem-solving methods, cell growth efficiency and antibody production can be improved. I hope this article can provide reference and help for your hybridoma cell culture experiments.
*Content generated by GPT-4 Turbo