CYTEK HLA-DR (L243) cFluor Anti-Human Instruction Manual

CYTEK HLA-DR (L243) cFluor Anti-Human

Instructions For Use

Copyright and Trademarks
© 2022 Cytek Biosciences, Inc. All rights reserved. Cytek, the Cytek logo, cFluor and Northern Lights are trademarks or registered trademarks of Cytek Biosciences, Inc. All other service marks, trademarks and tradenames are the property of their respective owners.

Cytek Biosciences, Inc
47215 Lakeview Blvd.
Fremont, CA 94538
USA
1.877.92.CYTEK (1.877.922.9835)
products@cytekbio.com cytekbio.com

Emergo Europe
Prinsessegracht 20
2514 AP The Hague Netherlands

Intended use

This product is intended for in vitro diagnostic use to identify human cells expressing HLA-DR antigen molecules in countries where the regulatory approval has been obtained from the local regulatory authorities.

Application

The L243 monoclonal antibody binds to the human HLA-DR antigen, a 36-kDa alpha heavy chain
and a 27-kDa beta light chain heterodimeric cell surface glycoprotein in transmembrane major
histocompatibility complex 2 (MHC II) family. HLA-DR, also known as human leukocyte antigen DR
isotype, is present on the surface of antigen-presenting cells, including B cells, dendritic cells,
macrophages, monocytes, and activated T cells. MHC class II regulates the immune system by
playing a critical role in binding and presenting antigen-derived peptides to peptide-MHC II-specific CD4 T cells. The antibody is conjugated to a fluorophore and purified by affinity chromatography.

Components

HLA-DR monoclonal antibody conjugated with the following listed cFluor fluorescent dye is
supplied in phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide and 0.2% BSA (BSA
Country of Origin USA).

Storage and Handling

This product is stable until the expiration date shown on the label when stored away from light at 2 ~ 8 ℃. Do not freeze.

Other Materials required but not supplied

• RBC lysing solution
• Pipettes and pipette tips of 20 μL, 100 μL and 1000 μL
• 12x75mm tube
• Vortex mixer
• Flow cytometer

Specimen Requirements

1. Require peripheral blood of not less than 500 μL collected by venipuncture in EDTA anticoagulation tube.
2. After collection, the samples should be stored at room temperature (18 ~ 25 ℃). Avoid shaking. The storage time should not exceed 24 hours.
3. After staining, the samples should be stored at 2 ~ 8 ℃ away from light and analyzed by flow cytometry within 2 hours.
4. Avoid samples with microbial contamination or coagulation.

Procedure

1. Add 100 μL well-mixed EDTA anticoagulated whole blood to the bottom of a tube. Avoid blood
   touching the upper tube wall.

2. Briefly centrifuge this product before use. Add 5 μL of HLA-DR-cFluor-conjugated reagent to the bottom of the tube.

3. Mix well by vortex and incubate for 15-30 minutes at room temperature and away from light.

4. Add 2 mL of 1 X lysis buffer into the tube, mix briefly by vortex, and incubate for 10-15 minutes at room temperature in the dark.

5. Centrifuge at 300g for 5 minutes, discard the supernatant, add 2 mL PBS with 0.02% BSA, and 0.09% NaN3 to resuspend the cell.

6. Centrifuge at 300g for 5 minutes, discard the supernatant, add 300 μL PBS with 0.02% BSA, and 0.09% NaN3 to resuspend the cells and keep at 4 ℃, and analyze on flow cytometer within 2 hours. If delayed analysis is needed (more than 2 hours), 300 μL of PBS containing 1% paraformaldehyde should be used to resuspend the cells and store the sample in a refrigerator at 2-8 ℃ away from light, but the storage time should not exceed 24 hours.

Quality Control

• Instrument QC: Use the manufacturer recommended controls according to the model of the
  flow cytometer.

• Refer to the instrument User’s Guide for instrument maintenance.

Warnings

• This reagent contains traces of sodium azide. Do not pipette by mouth.
• Use appropriate personal protective equipment per the safety data sheet when using this product.
• Follow biosafety practice in compliance with federal, state, and local regulations to handle all biological samples and materials in contact with them.
• Contact Cytek Support or refer to cytekbio.com for details on troubleshooting.

Performance Characteristics

Accuracy

Three replicate tubes were stained with HLA-DR-cFluor-conjugated reagent and analyzed on Cytek
Northern Lights™ flow cytometer. The percent HLA-DR+ lymphocytes results were within the control blood target value range provided by the manufacturer.

Intra-batch precision

Ten replicate tubes were stained with the same batch of HLA-DR-cFluor- conjugated reagent and
analyzed on Cytek Northern Lights™ flow cytometer. The CV of percent HLA-DR+ lymphocytes was calculated and was within the acceptance criteria.

Inter-batch precision

Three replicate tubes were stained with three batches of HLA-DR-cFluor- conjugated reagent and
analyzed on Cytek Northern Lights™ flow cytometer. The CV of percent HLA-DR+ lymphocytes was calculated and was within the acceptance criteria.

CV≤15%

Staining stability

Three replicate tubes were stained with the same batch of HLA-DR-cFluor- conjugated reagent and
analyzed on Cytek Northern Lights™ flow cytometer at these timepoints: within 2-hour (T0), 24-
hour, and 48-hour after staining. The percent HLA-DR+ lymphocytes at each time point were
compared to T0, and the mean relative difference was calculated and was within the acceptance criteria.

Limitations

1. This reagent can be used with a flow cytometer and is not recommended for fluorescence
   microscopy and immunohistochemistry.

2. This reagent is a fluorescent labeled product. It is easy to quench with extended light exposure
   and should be handled away from light.

3. If not following the lyse wash procedure described above, the reagent performance can be affected.

4. The results may be affected by improper storage of reagents, coagulation of specimens,
   improper storage of specimens, or incomplete lysis of red blood cells in the samples.

5. The test results of this reagent are for clinical reference only. Patient history, other laboratory tests, and treatment response should also be considered for diagnosis.

References

• Karman, A J et al. 1982. Proc Natl Accad Sci U S A. 79(19):6013-6017
• Shackelford DA, et al. 1982. Immunol Rev. 66:133-87
• Wallsend E, et al. 2008. J Boil Chem. 23;283(21):14717-2

1cFluor® B515 is equivalent to CF®488A manufactured and provided by Biotium, Inc. under an Agreement between Biotium and Cytek (LICENSEE). The manufacture, use, sale, offer for sale, or import of the product is covered by one or more of the patents or pending applications owned or licensed by Biotium. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.

References

• Full Spectrum Flow Cytometry Systems | Discover the Advantages | Cytek Biosciences

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