Description
Product Introduction
This product is a ready-to-use, methylcellulose-modified semi-solid medium designed for the screening of hybridoma cells. Under fluorescence microscopy, hybridoma cells secreting the target antibody form distinct fluorescent spots, whereas negative cells do not exhibit this feature. When used with a fluorescence stereomicroscope, it enables efficient screening of desired monoclonal clones, significantly saving time and reducing costs.
This semi-solid medium for rapid fluorescent hybridoma screening is pre-supplemented with essential nutrients to ensure rapid and stable cell growth within the semi-solid matrix. Hybridoma cells are seeded at low density to ensure adequate single-cell dispersion, and a fluorescently labeled antigen (provided by the user) is added for co-culture. After 3–5 days of incubation, individual cells proliferate to form distinct hybridoma clones. Positive clones secreting the specific target antibody generate a bright, specific fluorescent signal, while negative clones show no fluorescence. Furthermore, fluorescence intensity provides a visual indication of antibody secretion levels, facilitating the rapid selection of high-quality hybridoma cell lines with high expression and secretion capabilities.
The product contains methylcellulose, serum, the HAT (hypoxanthine-aminopterin-thymidine) selection system, penicillin-streptomycin antibiotics, phenol red indicator, and other key additives required for hybridoma proliferation.
Product features
Time- and labor-saving: Screening and cloning are completed in a single step, resulting in a shorter timeline and lower costs;
Epitope fidelity：Antigen-antibody binding occurs in a liquid-phase, semi-solid environment, preserving natural epitope conformation—superior to traditional solid-phase ELISA screening;
Visual tracking：Fluorescent signals allow for the direct localization of positive clones, with signal intensity indicating secretion levels, thereby preventing the loss of positive clones.
Instructions for Use
1. Prepare fluorescently labeled antigen and generate hybridoma cells (specific steps omitted here).
Note:
① Fluorescently labeled antigens must be provided by the user; common labels include fluorescent dyes such as 488 or FITC, or the target gene may be expressed as a fusion protein with a fluorescent protein (e.g., GFP) during recombinant antigen preparation.
② SP2/0 myeloma cells must be thawed and passaged at least one week prior to hybridoma fusion to ensure they are free of mycoplasma contamination and are in the logarithmic growth phase on the day of fusion.
③ Cells resulting from the fusion of SP2/0 and B lymphocytes are relatively fragile; therefore, the fused cells must first be placed in liquid medium containing cytokines and HAT, and incubated at 37°C in a 5% CO₂ incubator for 2–4 hours to facilitate recovery.
2. Semi-solid cloning and screening (example using a 6-well plate)
The semi-solid medium for rapid fluorescence screening of hybridomas must be thoroughly mixed before use.
For initial screening post-fusion
Calculate the cell density based on the amount of SP2/0 cells used during fusion. Add the semi-solid medium for rapid fluorescence-based hybridoma screening, the hybridoma cells, and the fluorescent antigen into the same well of a 6-well plate according to the quantities specified in Table 1; mix gently, and incubate undisturbed at 37°C in a 5% CO₂ incubator for 5 days.
Table 1
|
Reagent / Cell |
Amount |
|
Hybridoma Screening Fluorescence Semi-Solid Medium |
3 mL/well |
|
Hybridoma Cells |
1 × 10⁶–2 × 10⁶ cells/well |
|
Fluorescently Labeled Antigen |
Final concentration: 50–100 nM |
For subcloning and screening:
Add the hybridoma rapid fluorescence screening semi-solid medium, hybridoma cells, and fluorescent antigen to the same well of a 6-well plate according to the quantities specified in Table 2; mix gently, and incubate undisturbed at 37°C in a 5% CO₂ incubator for 5 days.
Table 2
|
Reagent / Cell |
Amount |
|
Hybridoma Screening Fluorescence Semi-Solid Medium |
3 mL/well |
|
Hybridoma Cells |
500 cells/well |
|
Fluorescently Labeled Antigen |
Final concentration: 50–100 nM |
Note:
① When setting up multiple wells (total of *n* wells), it is recommended to calculate the total reagent volume based on *n* + 1 wells. In a suitable container (e.g., a 50 mL centrifuge tube), add the fluorescent antigen to the rapid hybridoma fluorescence screening semi-solid medium to a final concentration of 50–100 nM; vortex to mix thoroughly, and centrifuge briefly (10 seconds) to remove air bubbles.
② Aliquot 3 mL into each well of a 6-well plate, then add the hybridoma cells and mix gently. Handle the cell culture plate gently and keep it level to prevent the displacement of cells and fluorescent signals within the semi-solid medium.
3. Clone Picking and Screening (Fluorescence stereomicroscope required)
(1) Observe the proliferation status and approximate number of positive clones (exhibiting fluorescence) under a fluorescence microscope;
(2) Prepare a 96-well plate and add 200 μL of liquid culture medium to each well;
(3) Using a fluorescence stereomicroscope and a 10 μL micropipette, aspirate cell clusters positive for the fluorescent signal (characterized by visible bright fluorescence in the surrounding area), transfer them into the 96-well plate, and incubate undisturbed at 37°C in a 5% CO₂ incubator for 2–5 days;
(4) Once the cells cover more than 30% of the well bottom, proceed with the following steps: ① ELISA detection; ② repeat subcloning; ③ scale-up culture (sequentially scaling up from 96-well plate → 24-well plate → 12-well plate → 6-well plate → 6 cm dish → 10 cm dish → shake flask).
Product Precautions
1. This product must be thoroughly mixed before use.
2. Strict aseptic techniques must be maintained throughout the experiment;
3. The laying of the board must be performed gently, minimizing the formation of air bubbles.
4. Maintain a high-humidity environment during incubation to prevent evaporation of the culture medium;
5. For your safety and health, please wear a lab coat and disposable gloves while working.
6. This product is for research use only.