The mouse monoclonal antibody M19H specifically binds to the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). CD19 antigen is a B-cell specific cell surface antigen, which is expressed in all B-cell lineage malignancies and normal B-cells. The scFv region of FMC63 has been used to develop CD19-specific chimeric antigen receptor (CAR) T cells utilized in clinical trials.
Item Information | |
Catalog # | 300401 |
Size/Concentration | 25 tests |
Price | $265.00 |
Specification |
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Host Species | Mouse |
Antibody Type | Monoclonal |
Antigen Purification | Protein A |
Fluorescent Dye | R-PE |
Excitation (nm) | 565 |
Reactivity | Mouse |
Storage Buffer | Aqueous buffered solution containing protein stabilizer and ≤0.05% ProClin 300. |
Application | FCM |
Preparation and Storage
Shipping | The product is shipped at 2-8°C. |
Stability & Storage | To provide optimal stability of reagent:
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Flow Cytometry FACS Protocol
- Reagents Required
- Phosphate Buffered Saline (PBS with a pH ~7.4).
- FACS Buffer (PBS containing 2% of BSA).
- Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647
- Preparation of Reagents
- Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647.
- The monoclonal Antibody must be diluted before use for FACS.
- Dilute the monoclonal antibody at a dilution of 1:100 in FACS buffer.
- Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647.
- Staining Protocol
- Harvest the cells from source and wash the cells once using FACS buffer.
- Perform a cell count and a cell viability count. Cell viability must be ≥ 95% with a cell number above 2×105 live cells.
- In a round-bottom test tube, resuspend cells in 100 µL of diluted Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647 for 30 min at 4°C.
- Wash the mixture of cells and antibody 3 times by centrifuge using FACS buffer.
- Resuspend the stained cells in 200 µL PBS per cell sample.
- Transfer the stained cell into a desired flow tube for analyzation on desired Flow Cytometer. The recommended acquisition rate is >10,000 events.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Antibody solutions containing ProClin 300 should be handled with care. Do not take internally and avoid all contact with the skin, mucosa and eyes.
Intellectual Product Notices
For Research Use Only. Not for use in diagnostic or therapeutic procedures
- This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer and the buyer must not use this product or its components:
for:- diagnostic, therapeutic or prophylactic purposes; or
- manufacturing or quality assurance or quality control.
- and/or to sell or transfer this product or its components for resale, whether or not resold for use in research.
- For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5781 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@thermofisher.com.
- The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. Bioswan will not be held responsible for patent infringement or other violations that may occur with the use of our products.
- Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product.
- Any use of this product other than the permitted use without the express written authorization of BioSwan Company is strictly prohibited.
Not for resales. BioSwan, the BioSwan Logo and all other trademarks are property of BioSwan Laboratories, Co., Ltd..
Flow Cytometry Protocol
Required Reagents
- Phosphate Buffered Saline (PBS, pH ~7.4).
- FACS Buffer: PBS supplemented with 2% Bovine Serum Albumin (BSA).
- Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647-conjugated.
Reagent Preparation
- Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647
- This antibody must be diluted prior to use in flow cytometry applications. Prepare a dilution of 1:100 in FACS buffer.
Staining Protocol
-
Cell Preparation
- Harvest cells from the culture or tissue source, performing a single wash with FACS buffer to remove media or other contaminants.
- Conduct a cell count and assess viability, ensuring that viability is at least 95% and the total live cell count exceeds 2×10^5.
-
Antibody Incubation
- Resuspend the cells in 100 µL of the diluted Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody, Alexa Fluor 647.
- Incubate for 30 minutes at 4°C, ensuring the tube is mixed gently but thoroughly to promote even antibody binding.
-
Washing
- Wash the cell-antibody mixture three times by centrifugation using FACS buffer. This step removes unbound antibodies, reducing background staining.
-
Resuspension
- Following the final wash, resuspend the stained cells in 200 µL of PBS. This step prepares the cells for analysis and helps in preserving the fluorescent signal.
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Flow Cytometry Analysis
- Transfer the stained cells into appropriate tubes for flow cytometry.
- Proceed with analysis on the flow cytometer, aiming for an acquisition rate of more than 10,000 events to ensure sufficient data for reliable analysis.
Notes
- The dilution ratio for the antibody may need adjustment based on the specific experimental setup and the expression level of the antigen of interest.
- Adequate washing steps are critical to remove excess antibody and reduce nonspecific staining, thereby improving the specificity and clarity of the flow cytometry data.
- The recommended cell viability and count thresholds are important to ensure that the data collected is representative and reliable, minimizing the impact of dead cells on the analysis.
Citations List
- Liu M, Liu J, Liang Z, Dai K, Gan J, Wang Q, Xu Y, Chen YH, Wan X. CAR-Macrophages and CAR-T Cells Synergistically Kill Tumor Cells In Vitro. Cells. 2022; 11(22):3692. https://doi.org/10.3390/cells11223692