User Guide for CYTEK models including: DOC-00492 20-Color AML Panel, DOC-00492, 20-Color AML Panel, AML Panel, Panel
Cytek 20-Color AML Panel, cFluor Reagent Kit (19C) | Cytek Biosciences
Cytek Biosciences-全光谱细胞分析与分选解决方案 Download the
File Info : application/pdf, 5 Pages, 223.02KB
DocumentDocumentSample Preparation (Bone Marrow Bulk Lyse) Guidelines for Cytek® 20-Color AML Panel Contents Introduction ...................................................................................................................................................................................................................... 1 Materials ............................................................................................................................................................................................................................. 2 Sample Preparation........................................................................................................................................................................................................ 2 Bulk-lysing Bone Marrow....................................................................................................................................................................................... 2 Preparing ViaDye Red Fixable Viability Dye.......................................................................................................................................................... 2 Protocol for Staining Bulk-lysed Bone Marrow in Tubes ............................................................................................................................... 3 Viability Reference Control ....................................................................................................................................................................................... 3 Single Color Reference Controls......................................................................................................................................................................... 3 Multicolor Sample..................................................................................................................................................................................................... 3 Cell Fixation in Tubes............................................................................................................................................................................................... 4 Introduction For anyone working with the Cytek® 20-Color AML Panel to prepare and acquire bone marrow cells in the Cytek® Northern LightsTM 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: 1. 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. 2. Import experiment template "Cytek® 20-Color AML Panel Template-ViaDyeTM Red" into your SpectroFlo® library module. 3. Refer to Cytek® 20-Color AML Panel Acquisition Protocol for a step-by-step guide for sample acquisition and analysis in SpectroFlo® software. * 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 bone marrow collected in Heparin tube. * For best results, resuspend cells in stain buffer after staining and analyze samples on a 3-laser Cytek Northern LightsTM system or Cytek Aurora system 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. 1 DOC-00492 Rev. A, Effective Date: 01/02/2023 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 BrightTM 780, eBioscienceTM (P/N 78-0198-42) · ViaDyeTM Red Fixable Viability Dye, Cytek Biosciences, R7-60008 · RBC Lysis Buffer, TonboTM, TNB-4300 · Cell strainer, Corning, 40 µm, 07-201-430 · PBS, pH7.4, Corning 21-040-CM · Stain Buffer (BSA), BD Biosciences, 554657 · Paraformaldehyde solution 4% in PBS, TonboTM, TNB-8222 · Cytek® FSPTM CompBeads, B7-10011 Sample Preparation Bulk-lysing Bone Marrow 1. Collect bone marrow into Heparin tubes* 2. Prepare a fresh working 1X RBC Lysis Buffer containing 0.1% Paraformaldehyde. For example, to make 50 mL add 5 mL of 10X RBC Lysis Buffer, 1.25 mL of 4% Paraformaldehyde, and 43.75 mL of deionized water. 3. Transfer 13 mL of room temperature 1X lysis solution (with 0.1% Paraformaldehyde) into a 15 mL conical tube 4. Transfer 1 mL of well mixed bone marrow to the tube containing 13 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 5 minutes 7. Gently aspirate the supernatant without disturbing the pellet 8. Vortex gently 9. Add 10 mL of room temperature 1X lysis solution (with 0.1% Paraformaldehyde) to the pellet, mix well 10. Repeat steps (5)-(8) 11. Add 10 mL Stain Buffer, mix well 12. Centrifuge at 300 x g, for 5 minutes 13. Gently aspirate the supernatant without disturbing the pellet 14. Vortex gently 15. Repeat steps (11)-(14) 16. Resuspend in proper volume of Stain Buffer, filter through 40-µm cell strainer, and count cells using the hematology analyzer or flow cytometer, adjust cell conc. to be around 10 x 106/mL in Stain Buffer * Please note that only bone marrow collected in Heparin tubes has been tested using this method. Preparing ViaDyeTM Red Fixable Viability Dye 1. Completely thaw DMSO 2. Add 100 L DMSO to the lyophilized ViaDyeTM 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 1:100 in PBS (=10 M working solution) Use the working solution at 5 L per test 2 DOC-00492 Rev. A, Effective Date: 01/02/2023 Protocol for Staining Bulk-lysed Bone Marrow in Tubes Plan on using ~400,000 cells for each Single Stain Reference Control (20 fluorescence, 1 Viability and 1 Unstained Control), and ~1 million cells for each Multicolor Sample NOTE: For AML MRD evaluation, using ~ 10 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 530 x g, 5 minutes at room temperature 5. Decant supernatant and blot on paper towel 6. Vortex thoroughly 7. Add 5 L of working solution ViaDyeTM Red Fixable Viability Dye to the cell pellet 8. Vortex thoroughly 9. Incubate for 25 minutes at room temperature, protected from light 10. Add 3 mL of Stain Buffer 11. Centrifuge at 530 x g, 5 minutes at room temperature 12. Decant supernatant and blot on paper towel 13. Vortex thoroughly 14. Resuspend in 300 L Stain Buffer or go to step (1) in "Cell Fixation in Tubes" on page 4 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" on page 5 to fix the samples in 1% paraformaldehyde 15. Acquire at medium or high flow rate within 2 hours post staining if cells are not fixed Single Color Reference Controls 1. Label a 12 x 75 mm tube for each Single Stain Reference Control 2. Add ~50 L of lysed cells or 1 drop of Cytek® FSPTM CompBeads to each Single Stain Reference Control tube NOTE: See Table 1 on page 4 for reference control type recommendations for each marker. 3. Add 5 L of appropriate monoclonal antibody 4. Vortex thoroughly 5. Incubate for 25 minutes at room temperature, protected from light 6. For single stained cells add 3 mL of Stain Buffer 7. Centrifuge at 530 x g, 5 minutes at room temperature 8. Decant and blot on paper towel 9. Vortex thoroughly 10. For single stain beads, wash twice by adding 2ml of stain buffer (or PBS contain 1% BSA), centrifuging (6 minutes at 600g), and aspirating the supernatant leaving approximately 50µl of supernatant in the tube each time. 11. Resuspend cells or beads in 300 L Stain Buffer or go to step (1) in "Cell Fixation in Tubes" on page 4 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 12. Acquire at medium or high 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 antibody cocktail according to the number of Multicolor samples. Add 5 L per test of each antibody 3 DOC-00492 Rev. A, Effective Date: 01/02/2023 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 L 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 L supernatant per test. 3. Add ~1 million cells to Multicolor Sample tube 4. Add PBS to complete the final volume to 3 mL 5. Centrifuge at 530 x g, 5 minutes at room temperature 6. Decant supernatant and blot on paper towel 7. Vortex thoroughly 8. Add 5 L of working solution ViaDyeTM Red Fixable Viability Dye to the cell pellet 9. Vortex thoroughly 10. Add the antibody cocktail prepared in step (2) 11. Vortex thoroughly 12. Incubate for 25 minutes at room temperature, protected from light 13. Add 3 mL of Stain Buffer 14. Centrifuge at 530 x g, 5 minutes at room temperature 15. Decant supernatant and blot on paper towel 16. Vortex thoroughly 17. Resuspend in 300 L Stain Buffer or go to step (1) in "Cell Fixation in Tubes" on page 4 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 18. 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. 1. Dilute 4% paraformaldehyde in PBS to make 1% paraformaldehyde solution 2. Add 300 L of 1% paraformaldehyde to 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 L Stain Buffer for Single Stain Reference Controls and 400 L for Multicolor Samples 10. Store at 4oC and acquire within 24 hours post fixation Table 1. Reference Control Type Recommendations for Single Color Reference Controls Laser Violet Blue Target CD16 CD14 HLA-DR CD4 CD11b CD19 CD7 Fluorochrome cFluor® V420 cFluor® V450 cFluor® V505 cFluor® V547 cFluor® V610 Super BrightTM 780 cFluor® B515 4 Recommended Control Type Cells or Beads Cells or Beads Cells or Beads Cells or Beads Cells Only Cells or Beads Cells or Beads DOC-00492 Rev. A, Effective Date: 01/02/2023 CD15 cFluor® B548 CD34 cFluor® BYG575 CD33 cFluor® BYG610 CD71 cFluor® BYG667 CD38 cFluor® B690 CD117 cFluor® BYG710 CD56 cFluor® BYG750 CD10 cFluor® BYG781 CD13 cFluor® R659 CD5 cFluor® R685 Red CD123 cFluor® R720 CD64 cFluor® R780 CD45 cFluor® R840 NOTE: Recommendations are for use with Cytek® FSPTM CompBeads only. Cells or Beads Cells Only Cells Only Cells Only Cells or Beads Cells or Beads Cells Only Cells or Beads Cells or Beads Cells Only Cells or Beads Cells or Beads Cells 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 BrightTM and eBioscienceTM 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 fixatives. "Cytek", "SpectroFlo", "Northern Lights", "FSP", "ViaDye" 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. 5 DOC-00492 Rev. A, Effective Date: 01/02/2023Microsoft Word for Microsoft 365