Isolation of T cells, in vitro activation, transduction, expansion, phenotyping, and functional analysis
CAR T cells are 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 T cells for research. It includes isolation of T cells, followed by activation, gene-transfer of the CAR construct, CAR T cell expansion, as well as phenotyping and analysis of the final CAR T cell product.
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.
Prepare the 10 mM CFSE stock solution in 1 mL of DMSO. Use CFSE at a final concentration of 1 µL by diluting the stock solution 1:10,000 in PBS.
Freshly prepare the following staining cocktails for each sample.
▲ Note: Store staining cocktails in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.
Table 1: Staining cocktail for immune cell composition antibody panel.
Table 2: Staining cocktail for transduction efficiency antibody panel.
|CAR DR||-||PE||to be defined|
|Buffer||-||Fill up to 100 µL with buffer|
The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, 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.
Table 3: Optimal surface density when working with purified T cells.
|Culture plate||Growth area||Max. working|
|Total T cell|
|T Cell TransAct|
to add per well
|96 well||0.31 cm2||0.2 mL||0.3×106||2 μL|
|48 well||1 cm2||1 mL||1×106||10 μL|
|24 well||2 cm2||2 mL||2×106||20 μL|
|12 well||4 cm2||4 mL||4×106||40 μL|
|6 well||10 cm2||5 mL||5×106||50 μL|
For CAR T cell phenotyping, e.g., to assess activation, differentiation, proliferation, and exhaustion of CAR T cells, contact firstname.lastname@example.org. All CAR T cell phenotyping panels are compatible with MACSQuant® Express Modes.
(Optional) Target cell labeling for killing assay
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 2).
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 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 or 50,000 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”.
|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)|
|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 (# 130-093-607). 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, refer to the application note "General instructions for MACSPlex Cytokine Kits" also 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” 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 please select Help> Info> expressModes within the MACSQuantify™ Software. The version number of the Express Mode package is increasing with each Express Mode updates. Make sure the MACSQuant Instrument contains an Express Mode package with at least the same or higher version number than the special protocol is marked with.
For results refer to figure 7 in section “Results”.
Table 4: Antibodies used for analysis of immune cell composition and transduction efficiency.
▲ Note: Antibodies can be replaced/included according to the respective needs. For additional antibodies refer to www.miltenyibiotec.com/antibodies.
▲ Note: For additional CAR T cell phenotyping panels, e.g., to assess activation, differentiation, proliferation, and exhaustion of T cells, refer to Miltenyi Biotec's pre-tested antibody panels (MBTPs) that allow for easy and accurate flow cytometry analysis. To ensure reliable and reproducible results, the panels are validated by in-house experts and their quality is frequently confirmed by customers. MBTPs provide a straight-forward gating strategy, staining protocols, and a list of all important reagents and materials.
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