CYTEK 20-Color AML Panel Instructions

: June 9, 2024
: CYTEK

Table of Contents

CYTEK 20-Color AML Panel
Introduction
Materials
Sample Preparation
Multicolor Sample
Documents / Resources
- Read User Manual Online (PDF format)
- Download This Manual (PDF format)

CYTEK 20-Color AML Panel

Introduction

For anyone working with the Cytek® 20-Color AML Panel to prepare and acquire bone marrow cells in Cytek® Northern LightTM or Aurora cytometer equipped with violet, blue and red lasers or higher, here are Cytek’s recommended sample preparation procedures*. These are 3 additional items to make your workflow easier:

Import the Cytek® 20-Color AML Panel Tags to the fluorescent tag lists in your SpectroFlo® Library section. If you already have existing tags in your library, delete them or overwrite them with the tags in this list.
Import experiment template “Cytek® 20-Color AML Panel Template” into SpectroFlo®.
Refer to Cytek® 20-Color AML Panel Acquisition Protocol for a step-by-step guide for sample acquisition and analysis in SpectroFlo®.

Please note that this kit is designed for research use only and is not for use in diagnostic or therapeutic procedures. The following method has only been tested in bone marrow collected in the Heparin tube.
For best results, resuspend cells in stain buffer after staining and analyze samples on Northern LightTM with 3 Lasers or Aurora within 2 hours post staining. Fixation with 1% paraformaldehyde following the procedure described in this protocol on page 4 can be performed if acquisition needs to be done at a later time, however, be aware of possible changes in the MFI for some antigens as well as quantitative differences compared to fresh samples in the enumeration of some populations.

Materials

Fresh Bone Marrow collected in Heparin tubes
Cytek® 20-Color AML Panel, cFluor® Reagent Kit (19C) (P/N R7-40009) and CD19 Monoclonal Antibody (SJ25C1), Super Bright™ 780, eBioscience™ (P/N 78-0198-42)
Cytek® RBC Lyse/Fix Solution 10X, R7-60010
PBS, pH7.4, Corning 21-040-CM
Cell strainer, Corning, 40 µm, 07-201-430
Stain Buffer (BSA), BD Biosciences, 554657
Paraformaldehyde solution 4% in PBS, Tonbo™, TNB-8222
Cytek® FSPTM CompBeads, B7-10011

Sample Preparation

Stain and lyse/fix Bone Marrow in Tubes

Collect bone marrow into Heparin tubes*
Filter through a 40-µm cell strainer, and count cells using the hematology analyzer or flow cytometer, adjust cell conc. around 10 x 106 cells/mL in Stain Buffer

NOTE: If the cell count is > 20 x 106 cells/mL, dilute to 10 x 106 cells/mL in Stain Buffer.

Plan on using ~400,000 cells for each Single Stain Reference Control (20 fluorescence, and 1 Unstained Control), and ~1 million cells for each Multicolor Sample
NOTE: F or AML MRD evaluation, using ~ 10 x 106 cells for each Multicolor Sample (do not need to dilute the samples).

Single Color Reference Controls

Label a 12 x 75 mm tube for each Single Stain Reference Control
Add ~50 μL of filtered bone marrow or 1 drop of Cytek® FSP™ CompBeads to each Single Stain Reference Control tube
NOTE: See Table 1 on page 4 for reference control type recommendations for each marker.
Add 5 μL of appropriate monoclonal antibody
Vortex thoroughly
Incubate for 20 minutes at room temperature, protected from light
For single stained cells add 2 mL 1X RBC Lyse/Fix solution
NOTE: Prepare 1X RBC Lyse/Fix Solution from 10X RBC Lyse/Fix solution with deionized water. For example, to make 50 mL add 5 mL of 10X RBC Lyse/Fix solution, and 45 mL of deionized water.
Vortex the tube briefly to mix
Incubate in the dark for 15 minutes at room temperature
Centrifuge at 530 x g, 5 minutes at room temperature
Decant and blot on paper towel
Vortex thoroughly to resuspend the cell pellet
Add 3 mL of stain buffer to the tube
Centrifuge at 530 x g, 5 minutes at room temperature
Decant and blot on paper towel
Vortex thoroughly to resuspend the cell pellet
For single stain beads, wash twice by adding 2 mL of stain buffer (or PBS contain 1% BSA), centrifuging (at 600 x g for 6 minutes), and aspirating the supernatant leaving approximately 50 µL of supernatant in the tube each time.
Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” on page 3 to fix the cells or beads in 1% paraformaldehyde
NOTE: If the samples need to be stored at 4oC for more than 2 hours prior to collecting data, follow the steps in “Cell Fixation in Tubes” on page 4 to fix the samples in 1% paraformaldehyde
Acquire at medium or high flow rate within 2 hours post staining if cells are not fixed

Multicolor Sample

Label a 12 x 75 mm tube for each Multicolor sample
Prepare antibody cocktail according to the number of Multicolor samples. Add 5 μL per test of each antibody.
NOTE: Prepare one extra test for the multicolor cocktail to take in account for any reagent loss in the process (ex. make multicolor cocktail for 6 tests if you have 5 multicolor samples to stain). Take 100 μL of the cocktail per multicolor sample and discard any leftover. Make antibody cocktails fresh each time before use and DO NOT re-use pre-made cocktails. Centrifuge the antibody cocktails at 8,000 -10,000 x g, 5 minutes at room temperature to avoid antibody aggregates. Take 100 μL supernatant per test.
Add ~100 μL of filtered bone marrow to Multicolor Sample tube
Vortex thoroughly
Incubate for 20 minutes at room temperature, protected from light
Add 2 mL 1X RBC Lyse/Fix solution
Vortex the tube briefly to mix
Incubate in the dark for 15 minutes at room temperature
Centrifuge at 530 x g, 5 minutes at room temperature
Decant and blot on paper towel
Vortex thoroughly to resuspend the cell pellet
Add 3 mL of stain buffer to the tube
Centrifuge at 530 x g, 5 minutes at room temperature
Decant and blot on paper towel
Vortex thoroughly to resuspend the cell pellet
Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” on page 3 to fix the cells in 1% paraformaldehyde
NOTE: If the samples need to be stored at 4oC for more than 2 hours prior to collecting data, follow the steps in “Cell Fixation in Tubes” on page 4 to fix the samples in 1% paraformaldehyde
Acquire at medium or high flow rate within 2 hours post staining if cells are not fixed

Cell Fixation in Tubes
If the samples need to be stored at 4oC for more than 2 hours prior to collecting data, follow these steps to fix the samples in 1% paraformaldehyde and acquire within 24 hours post-fixation.

Dilute 4% paraformaldehyde in PBS to make 1% paraformaldehyde solution
Add 300 μL of 1% paraformaldehyde to cell pellet.
Vortex thoroughly.
Incubate for 20 minutes at room temperature, protected from light
Add 3 mL of Stain Buffer
Centrifuge at 400 x g, 5 minutes at room temperature
Decant and blot on paper towel
Vortex thoroughly
Resuspend in 300 μL Stain Buffer for Single Stain Reference Controls and 400 μL for Multicolor Samples
Store at 4oC and acquire within 24 hours post-fixation

Table 1. Reference Control Type Recommendations for Single Color Reference Controls

Laser	Target	Fluorochrome	Recommended Control Type

Violet

Blue

Red

NOTE: Recommendations are for use with Cytek® FSPTM CompBeads only.

For Research Use Only. Not intended for use in diagnostic procedures.
cFluor® V547, cFluor® B515, cFluor® B548, cFluor® BYG610, cFluor® R685, cFluor® R720 and cFluor® R840 are equivalent to CF®405L, CF®488A, CF®514, PE- CF®596R, CF®660C, CF®700 and APC-CF®790T respectively, 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.
Super Bright™ and eBioscience™ are trademarks of Thermo Fisher Scientific. Cytek® FSP® CompBeads are developed and manufactured by Slingshot Biosciences, Inc. cFluor® BYG610, cFluor® BYG667, cFluor® BYG710, cFluor® BYG750, and cFluor® BYG781 are tandem dyes made with R-PE. cFluor® B690 is a tandem dye made with PerCP. cFluor® R780 and cFluor® R840 are tandem dyes made with APC. Caution – Tandem dyes may show changes in their emission spectra with prolonged exposure to light or fixative. “Cytek”, “SpectroFlo”, “Northern Lights”, “Full Spectrum Profiling”, “FSP” and “cFluor” are trademarks or registered trademarks of Cytek Biosciences, Inc. All other service marks, trademarks and tradenames appearing herein are the property of their respective owners.

DOC-00500 Rev A, Effective Date: 01/02/2023