Depletion of donor T cells, enrichment of donor NK cells, in vitro activation, transduction, expansion, phenotyping, and functional analysis.
CAR NK cells have to be developed, optimized, and validated in a pre-clinical setting by small-scale benchtop experiments. This application protocol describes a complete workflow for the engineering of CAR NK cells for research. It includes depletion of donor T cells, enrichment of donor NK cells, followed by activation, gene transfer of the CAR construct, and CAR NK cell expansion as well as the phenotyping and analysis of the final CAR NK cell product.
Supplement NK MACS Medium with 5% AB serum and MACS Cytokines Human IL-2, premium grade (500 U/mL), and Human IL-15, premium grade (140 U/mL).
▲Note: Make sure to freshly add Human IL-2, premium grade and Human IL-15, premium grade to the NK cell medium for effective NK cell expansion.
Prepare buffer by diluting the MACS BSA Stock Solution 1:20 with autoMACS® Rinsing Solution or PBS/EDTA.
▲ Note: Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
Freshly prepare the following master mixes for each sample.
▲ Note: Store master mix in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.
Table 1: Staining cocktail for NK cell count antibody panel.
Reagent | Volume (µL) |
---|---|
CD45-VioBlue® | 2 |
7-AAD Staining Solution | 10 |
FcR Blocking Reagent, human | 10 |
Buffer | 58 |
Table 2: Staining cocktail for NK cell composition antibody panel.
Reagent | Volume (µL) |
---|---|
CD45-VioBlue | 2 |
CD14-VioGreen™ | 2 |
TCRa/b Antibody-FITC | 2 |
CD3-PE | 5 |
CD56-APC | 2 |
CD19-APC-Vio® 770 | 2 |
7-AAD Staining Solution | 10 |
FcR Blocking Reagent, human | 10 |
Buffer | 65 |
Table 3: Staining cocktail for NK cell transduction antibody panel.
Reagent | Volume (µL) |
---|---|
CD45-VioBlue | 2 |
CD14-VioGreen | 2 |
Biotin Antibody-Vio Bright B515 | 2 |
CD3-PE | 5 |
CD56-APC | 2 |
7-AAD Staining Solution | 10 |
Buffer | 77 |
The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACDA, or citrate phosphate dextrose (CPD)). PBMCs should be isolated by density gradient centrifugation, e.g., using Ficoll-Paque™. Resuspend cell pellet in an appropriate amount of PBS-EDTA and proceed to magnetic labeling.
▲ Note: PBMCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding with magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS Cell Separation Reagents data sheets.
To detect the CAR NK cell rates, 20-50 µL aliquots were taken from the samples at several time points (day 1, 7, 10, 12, and 15) and stained with the NK cell count antibody panel (chapter 1, table 1).
On day 15, remove a small aliquot from the fraction representing the transduced CAR-expressing NK cells (e.g. 100–200 μL) for the three NK cell panels (chapter 1, tables 1–3).
▲ Note: Cell count, composition, and transduction efficiency can also be determined before and within the CAR NK cell expansion period.
For NK cell count panel:
For NK cell composition panel:
For NK cell transduction panel:
For determination of NK cell expansion rates samples of 20–50 µL are taken at appropriate time points (e.g. days 1, 7, 10, 12 and 15) and the count of viable cells is measured on the MACSQuant Analyzer 10. For dead cell exclusion propidium iodide is added prior to flow cytometric acquisition. The expansion rate is calculated afterwards.
For questions regarding flow cytometric analysis and phenotyping of CAR NK cells, contact macstec@miltenyi.com.
MACSPlex Assays are designed for determining concentrations of soluble analytes in a single sample. The analysis is based on MACSPlex Capture Beads, which display defined fluorescence properties and can be identified using standard flow cytometry techniques (figure 3).
The MACSPlex Cytokine 12 Kit, human is used to detect GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.
Setup of the co-cultures
Setup of the MACSPlex assay in a 96-well plate
Design the assay using two columns of the MACSPlex Filter Plate for the standards. Add each of the seven standard samples in duplicates next to each other. Standards should be run in order from the lowest concentration (blank control: 0 pg/mL) to the highest concentration (stock solution: 10,000 pg/mL). Start with the unknown sample in the next column of the plate. For more details, refer to the respective data sheet, section ”Protocol for the assay using the MACSPlex Filter Plate”.
Table 4: Setup of the assay using a 96-well plate.
Well position | Sample | Dilution |
---|---|---|
A1/A2 | Blank control | |
B1/B2 | MACSPlex Cytokine 12 Standard (3.2 pg/mL) | 1:3125 (Dilution5; 1:55) |
C1/C2 | MACSPlex Cytokine 12 Standard (16 pg/mL) | 1:625 (Dilution4; 1:54) |
D1/D2 | MACSPlex Cytokine 12 Standard (80 pg/mL) | 1:125 (Dilution3; 1:53) |
E1/E2 | MACSPlex Cytokine 12 Standard (400 pg/mL) | 1:25 (Dilution2; 1:52) |
F1/F2 | MACSPlex Cytokine 12 Standard (2 ng/mL) | 1:5 (Dilution1; 1:51) |
G1/G2 | MACSPlex Cytokine 12 Standard (10 ng/mL) | Stock solution (Dilution0; 1:50) |
H1/H2 | Leave empty | |
A3–H12 | Add unknown samples |
Flow cytometer setup
The kit includes MACSPlex Setup Beads for flow cytometer setup. MACSPlex Setup Beads are not required when using the MACSQuant Analyzer or MACSQuant Analyzer 10 but for all other flow instruments that can detect FITC, PE, and APC.
▲ Note: The kits are not suitable for use with the MACSQuant VYB.
Setup of the MACSQuant® Instrument
Calibrate the MACSQuant Instrument using MACSQuant Calibration Beads. For details, refer to the data sheet of the MACSQuant Calibration Beads. After successfully completing the calibration, the MACSQuant Instrument is ready for measurement. No further steps are required as all necessary setup steps are performed automatically during calibration.
Setup of other flow cytometers
The analysis of MACSPlex Cytokine 12 Kit, human requires a flow cytometer with a blue (e.g. 488 nm) and a red (e.g. 635 nm) laser, which can detect FITC, PE, and APC. For setting up these cytometers use MACSPlex Setup Beads (included in the kit). For instructions on the setup procedures of other flow cytometers, please refer to the application note " General instructions for MACSPlex Cytokine Kits: Data acquisition and analysis without the MACSQuant® Analyzer" available on the product page at www.miltenyibiotec.com/130-099-169 in the "Resources" section.
Flow cytometric acquisition and data analysis using the MACSQuant Express Mode
To perform the acquisition and data analysis of the MACSPlex Cytokine 12 Kit, human with the MACSQuant Instrument it is recommended to use the Express Modes MACSPlex_Standard and MACSPlex_Sample to achieve automated measurement and data analysis. For details refer to the special protocol “Data acquisition and analysis of the MACSPlex Cytokine Kits using the MACSQuant® Analyzer Express Modes in MACSQuantify™ Software version 2.13.1” available at www.miltenyibiotec.com/130-099-169 in the "Resources" section. The minimum version number of the Express Mode package needed to run the assay on the MACSQuant Instrument can be found there as well. To check the version number of the Express Mode package available on the MACSQuant Instrument in use please select Help> Info> expressModes within the MACSQuantify™ Software. The version number of the Express Mode package is increasing with each Express Mode update. Make sure the MACSQuant Instrument contains an Express Mode package with at least the same or a higher version number than the special protocol is marked with.
For results refer to figure 9 in section “Results”.
Table 5: Antibodies used for analysis of NK cell count, NK cell composition and NK cell transduction.
Antigen | Fluorochrome | Clone |
Biotin* | Vio Bright B515 | REA746 |
CD3 | PE | SK7 (BioLegend) |
CD14 | VioGreen | REA599 |
CD19 | APC-Vio 770 | REA675 |
CD45 | VioBlue | REA747 |
CD56 | APC | REA196 |
TCRa/b | FITC | REA652 |
7-AAD Staining Solution | - | - |
FcR Blocking Reagent, human | - | - |
* for biotinylated CAR Detection Reagent
▲ Note: Antibodies can be replaced/included according to the respective needs. For additional antibodies refer to www.miltenyibiotec.com/antibodies.
▲ Note: For additional CAR NK cell phenotyping panels, e.g., to assess activation, inhibition, and maturation of NK cells, contact macstec@miltenyi.com. All panels are compatible with MACSQuant Express Modes.
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