CYTEK R7-40002 25- Color Immunoprofiling Assay Instruction Manual

Sample Preparation (Whole Blood) Guidelines for Cytek® 25Color Immunoprofiling Assay

Introduction

For anyone working with the Cytek® 25-Color Immunoprofiling Assay to prepare and acquire whole blood cells in Cytek Aurora cytometer, here are Cytek’s recommended sample preparation procedures*. These are 3 additional items to make your workflow easier:

1. Import the Cytek® 25-Color Immunoprofiling Assay 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.
2. Import experiment template “Cytek® 25-Color IP Assay Template Whole Blood” into SpectroFlo®.

Refer to Cytek® 25-Color Immunoprofiling Assay 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. Following method has only been tested in blood collected in EDTA tube. * For best results, resuspend cells in stain buffer after staining and analyze samples on Aurora within 2 hours post staining. Fixation with 1% paraformaldehyde following the procedure described in this protocol in 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

• Whole blood collected in ETDA tubes
• Cytek® 25-Color Immunoprofiling Assay, cFluor® Reagent Kit (18C), Cytek Biosciences, R7-40002
• Immunoprofiling Kit, 7 Color (Brilliant Violet™), BioLegend, 900004160
• Brilliant Stain Buffer Plus, BD Biosciences, 566385Lysing Buffer (RUO), BD Biosciences, 555899
• PBS, pH7.4, Corning 21-040-CM
• Stain Buffer (BSA), BD Biosciences, 554657
• Paraformaldehyde solution 4% in PBS, Santa Cruz Biotechnology, Cat. sc-281692
• UltraComp eBeads™ Plus Compensation Beads, Thermo Fisher Scientific, 01-3333-4112 x 75 mm tubes or 96 well V-bottom deep plates (Corning 3960 or equivalent) and 96 well U-bottom polypropylene plates (Corning 3365 or equivalent)

Sample Preparation

Bulk-lysing Whole Blood

1. Collect whole blood into ETDA tubes*

2. Prepare a fresh working reagent of 1X BD Pharm Lyse Buffer by diluting 1:10 with deionized water.
   NOTE: Prepare fresh 1X lysis solution on the same day of the experiment.

3. Transfer 45 mL of room temperature 1X lysis solution into a 50 mL conical tube

4. Transfer 5 mL of well-mixed whole blood to the tube containing 45 mL of 1X lysis solution

5. Close and tighten the cap, mix gently by inverting or placing the tube on a tube rocker for 5 minutes

6. Centrifuge at 300 x g, for 8 minutes

7. Gently aspirate the supernatant without disturbing the pellet

8. Vortex gently

9. Add 45 mL of room temperature 1X lysis solution to the pellet, and mix well

10. Repeat steps (5)-(8)

11. Add 45 mL Stain Buffer, and mix well

12. Centrifuge at 300 x g, for 8 minutes

13. Gently aspirate the supernatant without disturbing the pellet

14. Vortex gently

15. Repeat steps (11)-(14)

16. Resuspend in half the volume of Stain Buffer as the original Whole Blood volume (ex) If you started with 5 mL of whole blood, resuspend in Stain Buffer to complete the final volume to 2.5 mL

• Please note that only blood collected in EDTA tubes has been tested using this method.

Protocol for Staining Bulk-lysed Whole Blood in Tubes

Plan on using ~100 μL whole blood for each Single Stain Reference Control (25 fluorescence and 1 Unstained Control), and ~400 μL whole blood for each Multicolor Sample*. Viability dye staining is usually not needed for fresh blood samples.

• The recommendations are in volume of original whole blood prior to bulk lysis.

Single Color Reference Controls

1. Label a 12 x 75 mm tube for each Single Stain Reference Control

2. Add ~50 μL of lysed cells to each Single Stain Reference Control tube
   NOTE: See Table 1 in page 4 for sample type recommendations for each marker.

3. Add 5 μL of appropriate monoclonal antibody

4. Vortex thoroughly

5. Incubate for 20 minutes at room temperature, protected from light

6. Add 3 mL of Stain Buffer

7. Centrifuge at 400 x g, 5 minutes at room temperature

8. Decant and blot on paper towel

9. Vortex thoroughly

10. Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 4 to fix the cells in 1% paraformaldehyde
    NOTE: If the samples need to be stored at 4º C for more than 2 hours prior to collecting data, follow the steps in “Cell Fixation in Tubes” in page 4 to fix the samples in 1% paraformaldehyde

11. Acquire at medium flow rate within 2 hours post staining if cells are not fixed

Multicolor Sample

1. Label a 12 x 75 mm tube for each Multicolor sample

2. Prepare the antibody cocktail in a 1.5 mL tube. For one Multicolor sample, first, add 10 μL of Brilliant Stain Buffer Plus, then 5 μL of 23 mAbs one by one, except for cFluor® BYG710 TCR γδ and Brilliant Violet 421™ CCR7.
   NOTE: cFluor® BYG710 TCR γδ and Brilliant Violet 421™ CCR7 need to be added separately. DO NOT add these antibodies to the cocktail mix.
   NOTE: Brilliant Stain Buffer must be added first to the cocktail.
   NOTE: Prepare one extra test for the multicolor cocktail to take in account any reagent loss in the process (ex. make a multicolor cocktail for 6 tests if you have 5 multicolor samples to stain). Take 125 μL of the cocktail per multicolor sample and discard any leftovers. Make antibody cocktails fresh each time before use and DO NOT re-use pre-made cocktails.

3. Add 200 μL of RBC lysed cells to the Multicolor Sample tube

4. Add 5 μL of cFluor® BYG710 TCR γδ and 5 μL of Brilliant Violet 421™ CCR7

5. Vortex thoroughly

6. Incubate for 10 minutes at room temperature, protected from light

7. Add the antibody cocktail prepared in step (2)

8. Vortex thoroughly

9. Incubate for 20 minutes at room temperature, protected from light

10. Add 3 mL of Stain Buffer

11. Centrifuge at 400 x g, 5 minutes at room temperature

12. Decant supernatant and blot on paper towel

13. Vortex thoroughly

14. Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 4 to fix the cells in 1% paraformaldehyde
    NOTE: If the samples need to be stored at 4º C 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

15.  Acquire at medium flow rate within 2 hours post staining if cells are not fixed

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

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

Table 1. Sample Type Recommendations for Single Color Reference Controls

cFluor® V547, cFluor® B515, cFluor® B532, cFluor® R668, and cFluor® R720 are equivalent to CF®405L, CF®488A, CF®503, CF®647, and CF®700 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.
Cytek dye cFluor® R840 and its conjugates were developed, manufactured, and commercialized by BioLegend, Inc. under the trademark APC/Fire810. Cytek dye cFluor® BYG610 was developed, manufactured, and commercialized by BioLegend, Inc. under the trademark PE-Dazzle 594™. ™ *Fluor conjugated antibody manufactured and supplied by BioLegend Inc.
Brilliant Violet™, Brilliant Violet 421™, Brilliant Violet 510™, Brilliant Violet 570™, Brilliant Violet 605™, Brilliant Violet 650™, Brilliant Violet 711™, and Brilliant Violet 785™ are trademarks of Sirigen Group Ltd.
“Cytek,” “SpectroFlo” 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-00299; Rev B; Effective Date: 09/28/2021

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