PerCP-Cy5.5 Conjugation Kit
PercP Cy5.5.5 conjugated Mouse anti Duck CD8α antibody, clone Du CD8-1 (MCA2479) conjugated using the YNX Rapid PerCP-Cy5.5 Antibody Conjugation Kit (LNK14) prior to the flow cytometric analysis of Mallard peripheral blood cells.
Image caption:
Gating strategy. EDTA-blood was stained with 4A12-APC, anti-chBAFF-R (2C4)-PE, anti-duCD4-FITC, anti-duCD8-PerCp, and anti-2G3-APC-Cy7 in a no-lyse no-wash single-step one-tube procedure and analyzed by flow cytometry. Shown is the gating strategy for the separation of thrombocytes and leukocytes (A), followed by doublet exclusion. (B) Leukocytes were classified as CD4 or CD8 single positive or CD4/CD8 double negative cells. (C) By means of their scatter properties, CD4/CD8 double negative cells were subdivided into granulocytes and monocytes. (D) CD8+ leukocytes were classified into 2C4-negative T cells and 2C4-positive B cells. Initial thrombocyte and leukocyte discrimination (A) was followed by doublet exclusion. Fluorescence-based gates were always followed by FSC/SSC gating. FSC/SSC gates were used to sort purify the respective cell populations (purity, >95%) and prepare cytospin slides followed by DiffQuick staining for thrombocytes (I), CD4+ T cells (II), granulocytes (III), monocytes (IV), CD8+ T cells (V), and B cells (VI).
From: Jax E, Werner E, Müller I, Schaerer B, Kohn M, Olofsson J, Waldenström J, Kraus RHS, Härtle S.
Evaluating Effects of AIV Infection Status on Ducks Using a Flow Cytometry-Based Differential Blood Count.
Microbiol Spectr. 2023 Jun 15:e0435122.
doi: 10.1128/spectrum.04351-22.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
LYNX Rapid PerCP-Cy5.5 Antibody Conjugation Kit (LNK14) used to conjugate fluorochrome to Mouse anti Chicken CD8 antibody prior to analysis of chicken blood cells.
Image caption:
Flow cytometry gating
EDTA-blood was stained with mix of anti-TCRαβ/Vβ1-FITC), anti-TCRαβ/Vβ2-FITC (clone TCR3), anti-TCRγδ-PE (clone TCR1), anti-Bu1-Pacific Blue (clone AV20), anti-CD8-PerCP-Cy5.5 (clone CT8, anti-CD45-APC (clone UM16-6,) and thrombocyte marker in a no-lyse no-wash onetube procedure and subsequently analyzed by flow cytometry.
We first separated thrombocytes and leukocytes (A) followed by a single cell gate (B and D). Leukocytes were subdivided in B cells (E), yd-T cells (G) and aß-T cells (J). T cell subpopulations were further separated in CD8pos and CD8neg cells (I and L). CD8neg aß-T cells were addressed as CD4pos T cells. To exclude potentially contaminating erythrocytes, for all cell populations an additional FSC/SSC gating was performed (C, F, H and K).
From: Sabsabi MA, Kheimar A, You Y, von La Roche D, Härtle S, Göbel TW, von Heyl T, Schusser B, Kaufer BB.
Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.
mBio. 2024 Aug 14;15(8):e0031524.
doi: 10.1128/mbio.00315-24.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
LYNX Rapid PerCP-Cy5.5 Antibody Conjugation Kit
- Product Type
- Conjugation Kit
- Specificity
- PerCP-Cy5.5 Conjugation Kit
Antibody Quality and Performance Guarantee| Product Code | Applications | Pack Size | List Price | Your Price | Qty | ||||||||||
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| LYNX Rapid PerCP-Cy5.5 Antibody Conjugation Kit® enables the rapid conjugation of a pre-prepared lyophilized mixture containing Peridinin Chlorophyll protein (PerCP)-Cy5.5 label to an antibody or protein. Activation of proprietary reagents within the antibody-label solution results in directional covalent bonding of PerCP-Cy5.5 to the antibody. The LYNX Rapid Conjugation kits® can be used to label small quantities of antibody/protein at near neutral pH, allowing a high conjugation efficiency with 100% antibody recovery. |
- Reagents In The Kit
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LNK144PERCPCY5.5: 3 Vials of 10ug LYNX lyophilized PerCP-Cy5.5 mix
1 Vial LYNX Modifier reagent
1 Vial LYNX Quencher reagent -
LNK142PERCPCY5.5: 3 Vials of 100ug LYNX lyophilized PerCP-Cy5.5 mix
1 Vial LYNX Modifier reagent
1 Vial LYNX Quencher reagent -
LNK141PERCPCY5.5: 1 Vial of 100ug LYNX lyophilized PerCP-Cy5.5 mix
1 Vial LYNX Modifier reagent
1 Vial LYNX Quencher reagent - Preparing The Antibody
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LNK144PERCPCY5.5
The following buffer solutions are recommended for preparing the antibody:
10-50mM amine-free buffer (e.g HEPES, MES, MOPS and phosphate) pH range 6.5-8.5, although moderate concentrations of Tris buffer (<20mM) may be tolerated.
Do not use buffers containing nucleophilic components e.g primary amines and thiols since they may react with LYNX chemicals. Azide (0.02-0.1%), EDTA and common non-buffering salts and sugars have little or no effect on conjugation efficiency.
The molar ratio of antibody: PerCP-Cy5.5 should be 1:1, i.e. 10ug antibody to every 10ug PerCP-Cy5.5. For optimal results the antibody should be at a concentration of 1mg/ml, with a maximum volume of 10ul and a maximum antibody amount of 10ug. Antibody at a concentration of greater than 1mg/ml requires dilution. Antibody below 1mg/ml can still be used as long as the maximum volume is not exceeded. Using less than the recommended amount of antibody may result in unbound label, but this will be removed during subsequent application wash steps. Antibody below 0.5mg/ml should be concentrated before use with the kit. -
LNK142PERCPCY5.5, LNK141PERCPCY5.5
The following buffer solutions are recommended for preparing the antibody:
10-50mM amine-free buffer (e.g HEPES, MES, MOPS and phosphate) pH range 6.5-8.5, although moderate concentrations of Tris buffer (<20mM) may be tolerated.
Do not use buffers containing nucleophilic components e.g primary amines and thiols since they may react with LYNX chemicals. Azide (0.02-0.1%), EDTA and common non-buffering salts and sugars have little or no effect on conjugation efficiency.
The molar ratio of antibody: PerCP-Cy5.5 should be 1:1, i.e. 100ug antibody to every 100ug PerCP-Cy5.5. For optimal results the antibody should be at a concentration of 1mg/ml, with a maximum volume of 100ul and a maximum antibody amount of 100ug. Antibody at a concentration of greater than 1mg/ml requires dilution. Antibody below 1mg/ml can still be used as long as the maximum volume is not exceeded. Using less than the recommended amount of antibody may result in unbound label, but this will be removed during subsequent application wash steps. Antibody below 0.5mg/ml should be concentrated before use with the kit. - Regulatory
- For research purposes only
- Guarantee
- 12 months from date of despatch
- Acknowledgements
- This product or portions thereof is manufactured under license from Carnegie Mellon University under U.S. Patent Number 5,268,486 and related patents. Cy and CyDye are trademarks of GE Healthcare Limited.
- Licensed Use
- These products and the methodology of conjugation are patent protected under United Kingdom patent number 2446088 and associated international patent applications. The purchase of this product conveys to the buyer the limited, non exclusive non-transferable right (without the right to resell repackage or further sublicense) under these patents to use the product to make conjugates for research and development purposes only. The purchaser cannot sell or otherwise transfer this product, or its components, or materials or data made using this product, or its components to a third party. Further information on purchasing licenses for diagnostic and other uses may be obtained by contacting Bio-Rad, at. Endeavour House, Langford Business Park, Langford Lane, Kidlington, Oxon. OX5 1GE UNITED KINGDOM. Tel: +44 1865 852 700. E-mail: antibodies@bio-rad.com
This kit contains lyophilized hygroscopic components that are moisture-sensitive. This kit is shipped under ambient conditions with silica packets to avoid exposure to moisture. On receipt, Bio-Rad recommend that the kit is stored at -20oC and protected from moisture. Storage in frost-free freezers is not recommended.This product should be stored undiluted. Avoid repeated freezing and thawing. Before opening, allow the components to reach room temperature to minimize condensation.
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
We recommend that for each conjugation the user determines the best antibody:conjugate ratio.
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| Conjugation |  |
We recommend that for each conjugation the user determines the best antibody:conjugate ratio.
- Instructions For Use
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LNK144PERCPCY5.5, LNK142PERCPCY5.5, LNK141PERCPCY5.5
1.To the antibody sample add 1ul of the Modifier reagent for every 10ul of antibody and mix gently.
2.Pipette the mixed antibody-modifier sample directly onto the LYNX lyophilized mix and gently pipette up and down twice to resuspend.
3.Replace cap onto vial and incubate in the dark at room temperature (20-25oC) for 3 hours, or overnight if preferred.
4.After incubation, add 1ul of Quencher reagent for every 10ul of antibody used. Leave to stand for 30 minutes before use.
References for PerCP-Cy5.5 Conjugation Kit
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Jamaly, S. et al. (2018) Elevated plasma levels of P-selectin glycoprotein ligand-1-positive microvesicles in patients with unprovoked venous thromboembolism.
J Thromb Haemost. May 31 [Epub ahead of print]. -
Jax, E. et al. (2023) Evaluating Effects of AIV Infection Status on Ducks Using a Flow Cytometry-Based Differential Blood Count.
Microbiol Spectr. : e0435122. -
Sabsabi, M.A. et al. (2024) Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.
mBio. 15 (8): e0031524.
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