ReadiUse™ 6-Color Human TBNK Antibody Kit Dry Reagent Format

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

The ReadiUse™ 6-Color Human TBNK Antibody Kit for flow cytometry utilizes dried multicolor beads to delineate principal lymphocyte subsets - CD4+ T cells, CD8+ T cells, B cells, and NK cells – in whole blood specimens. Each TBNK dried reagent contains a cocktail of fluorescently-labeled monoclonal antibodies directed against human CD3, CD4, CD8, CD16, C19, CD45, and CD56. The kit is designed to be used in a “lyse/no-wash” format for quick and easy sample preparation, with less inconsistencies. Also included in this kit is a viability stain to identify dead cells and a precise number of fluorescent counting beads to determine absolute lymphocyte subset cell concentrations.

Components

Example protocol

AT A GLANCE

Instrument

ReadiUse™ 6-Color Human TBNK Antibody Kit is designed to be used on a flow cytometer equipped with appropriate computer hardware and software. The flow cytometer must be equipped with:

488 nm Blue Laser and 635 nm Red Laser
Capable of detecting light scatter (forward and side)
Four-color fluorescence channels when excited by the 488nm laser: FITC, PE, PerCP-Cy5.5, & PE-Cy7
Two-color fluorescence channels when excited by the 635nm laser: APC & APC-Cy7
Optional: 405nm Violet Laser and equipped with 455nm detection channels

Specifications

Laser	Emission	Conjugate	Suggested Channel (Example: Cytek Aurora)
Blue	520 nm	CD3 (SK7) - iFluor™ 488	B2 (518-529 nm)
Blue	575 nm	CD16 (3G8) - PE, CD56 (MY31) - PE	B4 (560-583 nm)
Blue	700 nm	CD45 (2D1) - PerCP-iFluor™ 680	B9 (688-707 nm)
Blue	780 nm	CD4 (SK3) - PE-iFluor™ 750	B13 (772-795 nm)
Red	665 nm	CD19 (SJ25C1) - APC	R1 (652-669 nm)
Red	780 nm	CD8 (SK1) - APC-iFluor™ 750	R7 (772-795 nm)
Violet	455 nm	NucPO-1	V3 (451-466 nm)

Precautions

The reagent contains sodium azide. Sodium azide is harmful if swallowed (R22). Wear suitable protective clothing. If swallowed, seek medical advice immediately. Contact with acids liberates toxic gas. Azides should be flushed with large amounts of water during disposal to avoid deposits in lead or copper plumbing
The 10X Lyse/Fix Buffer (Component B) contains formaldehyde. Formaldehyde is toxic and suspected to be a carcinogen. Avoid inhalation or contact.
All blood specimens are considered biohazards. Handle them as if they are capable of transmitting infection and dispose of them with proper precautions and in accordance with governmental regulations.

PREPARATION OF WORKING SOLUTION

1X Lysis/Fix Buffer

Prepare the 1X Lysis/Fix Buffer by adding 3.0 mL of deionized water to one bottle of the 10X Lyse/Fix Buffer (Component B). Mix and relabel the bottle as 1X Lysis/Fix Buffer.

SAMPLE EXPERIMENTAL PROTOCOL

Sample Preparation

Prepare blood cell samples, either fresh blood collected in an EDTA tube or isolated PBMCs at a concentration no greater than 10⁶ cells/mL can be used.
Remove the desired number of reagent tubes from the pouch and reseal the pouch.
Thoroughly mix the sample and dispense exactly 50 µL of sample into the designated reagent tube. Note the accuracy of the sample dispense will affect the accuracy of the absolute cell concentration determined.
Gently vortex each tube for 30 seconds to ensure complete solubilization of the dried reagent.
Incubate for 20 minutes at room temperature. Protect the tube from direct light.
Add 450 µL of 1X Lysis/Fix Buffer to each tube and vortex for 10 seconds. Return tubes to the dark for at least 15 minutes.
Vortex sample tube thoroughly at low speeds and load onto cytometer for analysis. The ReadiUse™ 6-Color Human TBNK Antibody Kit is designed to be used in a Lyse/No-wash format. If the sample is washed before analysis, the ability to determine absolute cell concentrations will be lost.
Flow Cytometer Acquisition: start and operate flow cytometer according to manufacturer’s instructions. Adjust the threshold to minimize debris and ensure populations of interest are included. Analyze the data using the appropriate cytometer-specific software.

Images

Figure 1. Human peripheral blood was stained with the ReadiUse™ Human TBNK 6 Color Antibody Kit *Dry Reagent*. Cells were stained at room temperature for 20 minutes, lysed with 1X Lysis/Fix buffer for 15 minutes, and then analyzed by flow cytometry. Live cells were gated on NucPO-1 viability stain. Live cells are gated on CD45+ (2D1)-PerCP-iFluor® 680 for lymphocytes and then gated on CD3+ or CD3- (SK7) iFluor® 488 cell populations. CD3+ cells are shown using CD4 (SK3) PE-iFluor® 750 and CD8 (SK1) APC-iFluor® 750 markers. CD3- cells are shown using CD19 (SJ25C1)-APC and CD56 (5.1H11) / CD16 (B73.1)-PE markers.