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:

1. 488 nm Blue Laser and 635 nm Red Laser
2. Capable of detecting light scatter (forward and side)
3. Four-color fluorescence channels when excited by the 488nm laser: FITC, PE, PerCP-Cy5.5, & PE-Cy7
4. Two-color fluorescence channels when excited by the 635nm laser: APC & APC-Cy7
5. 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

1. 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
2. The 10X Lyse/Fix Buffer (Component B) contains formaldehyde. Formaldehyde is toxic and suspected to be a carcinogen. Avoid inhalation or contact.
3. 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

1. 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.
2. Remove the desired number of reagent tubes from the pouch and reseal the pouch.
3. 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.
4. Gently vortex each tube for 30 seconds to ensure complete solubilization of the dried reagent.
5. Incubate for 20 minutes at room temperature. Protect the tube from direct light.
6. 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.
7. 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.
8. 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.