Description
Product Introduction
This product is a ready-to-use, methylcellulose-supplemented medium designed for the semi-solid screening of hybridoma cells. During semi-solid culture, individual cells are immobilized within the viscous medium and proliferate to form distinct, independent monoclonal colonies. Picking these discrete colonies allows for the isolation of diverse monoclonal cell lines.
In hybridoma screening, this product increases monoclonal yields, simplifies the screening workflow, and shortens the screening period. It also reduces the consumption of culture media and plates, offering an alternative to the labor-intensive procedures of the traditional limiting dilution method.
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
High-efficiency cloning: enables single cells to form colonies *in situ*, rapidly yielding large numbers of monoclonal strains, reducing the risk of polyclonal contamination, and increasing monoclonal yield;
High compatibility：accommodates monoclonal strains with varying growth rates, reduces the loss rate of slower-growing positive clones, and increases the probability of identifying high-quality candidate strains;
Saves time and effort：replaces the traditional limiting dilution method, streamlines the screening process, and significantly shortens the experimental cycle.
Instructions for Use
1. Preparation of hybridoma cells (specific steps omitted here).
Note:
① At least one week prior to hybridoma fusion, thaw and subculture SP2/0 myeloma cells to ensure the absence of *Mycoplasma* contamination and confirm that the cells are in the logarithmic growth phase on the day of fusion.
② As the fused cells resulting from the fusion of SP2/0 and B lymphocytes are relatively fragile, they must first be incubated in liquid medium supplemented with cytokines and HAT in a 37°C, 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.
Used for initial screening after fusion
Calculate the cell density based on the amount of SP2/0 cells used during fusion; add the hybridoma rapid screening semi-solid medium and the hybridoma cells to 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 |
|
Viral transduction screening optically transparent solid culture medium |
3 mL/well |
|
Hybridoma Cells |
1 × 10⁶ cells/well |
For subcloning screening:
Add the hybridoma rapid screening semi-solid medium and hybridoma cells 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 |
|
Viral transduction screening optically transparent solid culture medium |
3 mL/well |
|
Hybridoma Cells |
500 cells/well |
Note:
① If seeding multiple wells (total of n wells), it is recommended to calculate the total volume of culture medium based on n+1 wells; dispense 3 mL into each well of a 6-well plate, then add the hybridoma cells and mix gently.
② Handle cell culture plates gently and keep them level to prevent the cells from shifting within the semi-solid medium.
3. Clone Picking and Screening
(1) Observe the proliferation status and approximate number of the cell clusters;
(2) Prepare a 96-well plate and add 200 μL of liquid medium to each well;
(3) Use a 10 μL micropipette to aspirate the cell clusters, transfer them to a 96-well plate, and incubate at 37°C in a 5% CO₂ incubator for 5–10 days.
Note: Clones of varying sizes should be selected; slow-growing or smaller clones may still possess excellent antibody-secreting capabilities. In traditional proliferation environments containing mixed polyclonal populations, such clones—being at a competitive disadvantage—are easily suppressed and lost to dominant clones, leading to the failure to detect high-quality candidate strains.
(4) Once the cells cover more than 30% of the well bottom, positive clones can be screened using appropriate methods (such as ELISA, flow cytometry, or Western blot).
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.