CYTEK 25-Color Immunoprofiling Assay Instructions

CYTEK 25-Color Immunoprofiling Assay

Introduction

For anyone working with Cytek® 25-Color Immunoprofiling Assay to prepare and acquire peripheral blood mononuclear cells (PBMCs) on Cytek Aurora cytometer, we recommend the following sample preparation instructions. 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 Cytek 25-Color IP Assay Template PBMC into SpectroFlo®.
3. 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.

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

• Frozen PBMCs
• ViaDye Red Fixable Viability Dye, Cytek Biosciences, R7-60008
• 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, 566385
• PBS, pH7.4, Corning 21-040-CM
• Stain Buffer (BSA), BD Biosciences, 554657
• Paraformaldehyde solution 4% in PBS, Santa Cruz Biotechnology, Cat. sc-281692
• 12 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

Thawing PBMCs

1. Pre-warm ~50 mL RPMI (10% FBS, 1% Penicillin) at 37°C for at least 30 minutes
2. Thaw PBMC vial quickly in 37oC water bath until the core is loose
3. Transfer the cells into a 50 mL conical tube
4. Add 1 mL of warm media to the empty cryovial. Set it aside.
5. Drop by drop, slowly add 10 mL of warm media to the cells in the 50 mL conical tube while gently swirling the tube to mix
6. Pour the content of the cryovial from step (4) into the 50 mL conical tube
7. Add additional media to complete the final volume to 20 mL
8. Centrifuge at 300 x g, 8 minutes
9. Decant the supernatant and blot on paper towel
10. Gently resuspend the pellet in 2 ml of warm media by pipetting up and down using a serological pipet
11. Repeat steps (7)-(10)
12. Resuspend in proper volume of warm media and count cells
13. Loosen the cap on the 50 mL conical tube, place the cells in the cell culture incubator until ready to use

Preparing ViaDye Red Fixable Viability Dye

1. Completely thaw DMSO

2. Add 100 μL DMSO to the lyophilized ViaDye Red Fixable Viability Dye stock (=1 mM stock solution)

3. Vortex to mix thoroughly

4. Aliquot and freeze at -20°C until use

5. Thaw an aliquot of the stock solution at room temperature, protected from light, before each use.
   NOTE : Do not re-freeze or re-use the viability dye

6. Dilute the stock solution at 1:500 in PBS (=2 μM working solution)

7. Use the working solution at 5 μL per test

Protocol for Staining PBMCs in Tubes

Plan on using ~400,000 cells for each Single Stain Reference Control (25 fluorescence, 1 Viability and 1 Unstained Control), and ~3 million cells for each Multicolor Sample.

Viability Reference Control

1. Label a 12 x 75 mm tube for Viability Reference Control

2. Add ~400,000 cells to the tube

3. Add PBS to complete the final volume to 3 mL

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

5. Decant supernatant and blot on paper towel

6. Vortex thoroughly

7. Repeat steps (3)-(6) if the volume in step (2) is bigger than 1 mL

8. Add 5 μL of working solution ViaDye Red Fixable Viability Dye to the cell pellet

9. Vortex thoroughly

10. Incubate for 15 minutes at room temperature, protected from light

11. Add 3 mL of Stain Buffer

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

13. Decant supernatant and blot on paper towel

14. Vortex thoroughly

15. Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 5 to fix the cells in 1%paraformaldehyde
    NOTE : If the samples need to be stored at 4oC for more than 2 hour prior to collecting data, follow the steps in “Cell Fixation in Tubes” in page 5 to fix the samples in 1% paraformaldehyde

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

Single Stain Reference Controls

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

2. Add ~400,000 cells to each tube

NOTE : See Table 1 in page 4 for sample type recommendations for each marker.

3. Add Stain Buffer to complete the final volume to 3 mL

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

5. Decant supernatant and blot on paper towel

6. Vortex thoroughly

7. Repeat steps (3)-(6) if the volume in step (2) is bigger than 1 mL

8. Add 5 μL of appropriate monoclonal antibody to the cell pellet

9. Vortex thoroughly

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

11. Add 3 mL of Stain Buffer

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

13. Decant and blot on paper towel

14. Vortex thoroughly

15. Resuspend in 300 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 5 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” in page 5 to fix the samples in 1% paraformaldehyde

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

Table 1. Sample Type Recommendations

Multicolor Sample

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

2. Prepare 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 in 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 for any reagent loss in the process (ex. make 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 leftover. Make antibody cocktails fresh each time before use and DO NOT re-use pre-made cocktails.

3. Add ~3 million cells to Multicolor Sample tube

4. Add PBS to complete the final volume to 3 mL

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

6. Decant supernatant and blot on paper towel

7. Vortex thoroughly

8. Repeat steps (4)-(7) if the volume in step (3) is bigger than 1 mL

9. Add 5 μL of working solution ViaDye Red Fixable Viability Dye to the cell pellet

10. Vortex thoroughly

11. Incubate for 15 minutes at room temperature, protected from light

12. Add 3 mL of Stain Buffer

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

14. Decant and blot on paper towel

15. Vortex thoroughly

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

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

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

19. Vortex thoroughly

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

21. Add 3 mL of Stain Buffer

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

23. Decant supernatant and blot on paper towel

24. Vortex thoroughly

25. Resuspend in 400 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 5 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” in page 5 to fix the samples in 1% paraformaldehyde

26. 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 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 of 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 4oC and acquire within 24 hours post fixation

Protocol for Staining PBMCs in 96 well Plates

Plan on using ~400,000 cells for each Single Stain Reference Control (25 fluorescence, 1 Viability and 1 Unstained Control), and ~3 million cells for each Multicolor Sample. Prepare separate plates for Single Stain Reference Controls and Multicolor Samples. Use a 96 deep well V-bottom plate (polystyrene or polypropylene) to prepare the cells and transfer the final sample to a 96 well U-bottom plate (polypropylene) for acquisition.

Viability Reference Control

1. Using a 96 deep well V-bottom plate, add ~400,000 cells to Viability Reference Control well

2. Add PBS to complete the final volume to 2 mL

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

4. Decant supernatant and blot on paper towel

5. Vortex thoroughly

6. Repeat steps (2)-(5) if the volume in step (1) is bigger than 1 mL

7. Add 5 μL of working solution ViaDye Red Fixable Viability Dye to the cell pellet

8. Mix well by pipetting up and down

9. Incubate for 15 minutes at room temperature, protected from light

10. Add Stain buffer to complete the final volume to 2 mL

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 200 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 7 to fix the cells in 1%paraformaldehyde
    NOTE : If the samples need to be stored at 4oC for more than 1 hour prior to collecting data, follow the steps in “Cell Fixation in Tubes” in page 7 to fix the samples in 1% paraformaldehyde

15. Transfer the sample to 96 well U-bottom polypropylene plate

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

Single Stain Reference Control

1. Using a 96 deep well V-bottom plate, Add ~400,000 cells to each Single Stain Reference Control well NOTE: See Table 1 in page 4 for sample type recommendations for each marker.

2. Add Stain Buffer to complete the final volume to 2 mL

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

4. Decant and blot on paper towel

5. Vortex thoroughly

6. Repeat steps (2)-(5) if the volume in step (1) is bigger than 1 mL

7. Add 5 μL of Mab to the cell pellet in each well

8. Mix well by pipetting up and down

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

10. Add Stain Buffer to complete the final volume to 2 mL per well

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

12. Decant and blot on paper towel

13. Vortex thoroughly

14. Resuspend in 200 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 7 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” in page 7 to fix the samples in 1% paraformaldehyde

15. Transfer the sample to 96 well U-bottom polypropylene plate

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

Multicolor Sample

1. Prepare 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 in 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 for any reagent loss in the process (ex. make 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 leftover. Make antibody cocktails fresh each time before use and DO NOT re-use pre-made cocktails.

2. Using a 96 deep well V-bottom plate, add ~3 million cells to Multicolor Sample well

3. Add PBS to complete the final volume to 2 mL

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

5. Decant and blot on paper towel

6. Vortex thoroughly

7. Repeat steps (3)-(6) if the volume in step (2) is bigger than 1 mL

8. Vortex thoroughly

9. Add 5 μL of working solution ViaDye Red Fixable Viability Dye to the cell pellet

10. Mix well by pipetting up and down

11. Incubate for 15 minutes at room temperature, protected from light

12. Add Stain Buffer to complete the final volume to 2 mL

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

14. Decant and blot on paper towel

15. Vortex thoroughly

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

17. Mix well by pipetting up and down

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

19. Add the antibody cocktail prepared in step (1)

20. Mix well by pipetting up and down

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

22. Add Stain Buffer to complete the final volume to 2 mL

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

24. Decant and blot on paper towel

25. Vortex thoroughly

26. Resuspend in 200 μL Stain Buffer or go to step (1) in “Cell Fixation in Tubes” in page 7 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” in page 7 to fix the samples in 1% paraformaldehyde

27. Transfer the sample to 96 well U-bottom polypropylene plate

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

Cell Fixation in Plates
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.

1. Dilute 4% paraformaldehyde in PBS to make 1% paraformaldehyde solution
2. Add 300 μL of 1% paraformaldehyde to cell pellet in each well.
3. Mix well by pipetting up and down
4. Incubate for 20 minutes at room temperature, protected from light
5. Add Stain Buffer to complete the final volume to 2 mL
6. Centrifuge at 400 x g, 5 minutes at room temperature
7. Decant and blot on paper towel
8. Vortex thoroughly
9. Resuspend in 200 μL Stain Buffer
10. Transfer the sample to 96 well U-bottom polypropylene plate
11. Store at 4oC and acquire within 24 hours post fixation

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 is commercialized by BioLegend, Inc. under the trademark APC/Fire810™. Cytek dye cFluor® BYG610 was developed, manufactured, and is commercialized by BioLegend, Inc. under the trademark PE-Dazzle 594™. *Fluor conjugated antibody manufactured and supplied by BioLegend Inc. 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-00298;  Rev B; Effective Date: 09/28/2021

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