Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.
Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.
Stem cell and chimeric antigen receptor (CAR) T-cell therapies are gaining popularity and are under active investigation for the treatment of various diseases, such as myocardial failure1,2, retinal degeneration2, macular degeneration2, diabetes2, myocardial infarction3,4,5, and cancers6,7,8,9,10. Among the two plausible approaches of stem cell therapies, stem cells can either be directly engrafted on the disease site to cause a therapeutic response, or cause changes in the microenvironment of the disease site without adhering to the disease site to initiate an indirect therapeutic response. An indirect therapeutic response could cause changes in the microenvironment of the disease site by releasing factors that would repair or treat the disease5. These approaches of stem cell therapies could be evaluated by noninvasive imaging of radiolabeled stem cells. Noninvasive imaging could correlate the uptake of the radiolabeled cells on the disease site with a therapeutic response to decipher the direct versus indirect therapeutic response.
Additionally, immune cell-based therapies are being developed to treat various cancers using CAR T-cell6,7,8,9,10and dendritic cell immunotherapy11,12. Mechanistically, in CAR T-cell immunotherapy6,7,8,9,10, T-cells are engineered to express an epitope that binds to a specific antigen on tumors that needs to be treated. These engineered CAR T-cells, upon administration, bind to the specific antigen present on the tumor cells through an epitope-antigen interaction. After binding, the bound CAR T-cells undergo activation and then proliferate and release cytokines, which signals the immune system of the host to attack the tumor expressing the specific antigen. In contrast, in the case of dendritic cell therapies11,12, dendritic cells are engineered to present a specific cancer antigen on their surface. These engineered dendritic cells, when administered, home to the lymph nodes and bind to the T-cells in the lymph nodes. The T-cells, upon binding to the specific cancer antigens on the administered dendritic cells, undergo activation/proliferation and initiate an immune response of the host against the tumor expressing that specific antigen. Hence, the assessment of trafficking of administered CAR T-cells to a tumor site9,10 and homing of dendritic cells to the lymph nodes11,12 is possible by imaging radiolabeled CAR T-cells and dendritic cells to determine the efficacy of immunotherapy. Furthermore, noninvasive cell trafficking can help to better understand the therapeutic potential, clarify the direct versus indirect therapeutic response, and predict and monitor the therapeutic response of both stem cell and immune cell-based therapies.
Different imaging modalities for cell trafficking have been explored3,4,9,10,12, including optical imaging, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Each of these techniques has its own advantages and disadvantages. Among these, PET is the most promising modality due to its quantitative nature and high sensitivity, which are essential for the reliable quantification of cells in imaging-based cell trafficking3,4,9,10.
The positron-emitting radioisotope 89Zr, with a half-life of 78.4 h, is suitable for cell labeling. It allows PET imaging of cell trafficking for over 1 week and is readily produced by widely available, low-energy medical cyclotrons13,14,15,16,17. Additionally, an appropriately functionalized, p-isothiocyanatobenzyl-desferrioxamine (DFO-Bn-NCS) chelator is commercially available for the synthesis of a 89Zr-labeled, ready-to-use, cell labeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine, also known as [89Zr]Zr-DBN18,19,20,21,22,23,24,25. The principle of [89Zr]Zr-DBN-mediated cell labeling is based on a reaction between primary amines of cell membrane proteins and the isothiocyanate (NCS) moiety of [89Zr]Zr-DBN to produce a stable covalent thiourea bond.
[89Zr]Zr-DBN-based cell labeling and imaging have been published to track a variety of different cells, including stem cells18,23,25, dendritic cells18, cardiopoietic stem cells19, decidual stromal cells20, bone marrow-derived macrophages20, peripheral blood mononuclear cells20, Jurkat/CAR T-cells21, hepatocytes22,24, and white blood cells25. The following protocol provides step-by-step methods of preparation and cell radiolabeling with [89Zr]Zr-DBN and describes changes that may be required in the radiolabeling protocol for a specific cell type. For greater clarity, the method of cell radiolabeling presented here is divided into four sections. The first section deals with the preparation of [89Zr]Zr-DBN by chelating 89Zr with DFO-Bn-NCS. The second section describes the preparation of a biocompatible formulation of [89Zr]Zr-DBN that can be readily used for cell radiolabeling. The third section covers the steps needed for the preconditioning of cells for radiolabeling. The preconditioning of cells involves washing the cells with protein-free phosphate buffered saline (PBS) and HEPES buffered Hanks balanced salt solution (H-HBSS) to remove external proteins, which might interfere or compete with the reaction of [89Zr]Zr-DBN with primary amines present on the cell surface proteins during radiolabeling. The final section provides steps involved in the actual radiolabeling of the cells and quality control analysis.
Dendritic cells and melanoma cells were obtained commercially18. Hepatocytes were isolated from the liver of pigs following laparoscopic partial hepatectomy22,24. Stem cells were isolated from bone marrow aspirates18,19,26. The adipose tissue-derived stem cells were obtained from the Human Cellular Therapy Laboratory, Mayo Clinic Rochester23. Human white blood cells were isolated from the collected blood received from the Division of Transfusion Medicine, Mayo Clinic Rochester25. Various cells used for radiolabeling were obtained and used in compliance with guidelines recommended by the Institutional Animal Care and Use Committee, Mayo Clinic Stem Cell Research Oversight Subcommittee, Division of Transfusion Medicine Research Committee, Institutional Biosafety Committee, and by the Radiation Safety Committee.
1. Preparation of [ 89Zr ]Zr-p-isothiocyanatobenzyl-desferrioxamine ([ 89Zr ]Zr-DBN)
Timing: ~160-220 min
NOTE: For the preparation of [89Zr]Zr-DBN, isolate 89Zr in the form of [89Zr]Zr-hydrogen phosphate ([89Zr]Zr(HPO4)2) or [89Zr]Zr-chloride ([89Zr]ZrCl4), as mentioned in step 1.1.
Figure 1: Schematic of [89Zr]Zr-DBN preparation. For the preparation of [89Zr]Zr-DBN, neutralize preformulated [89Zr]Zr(HPO4)2 or [89Zr]ZrCl4 to a pH of 7.5-8.0. Incubate the neutralized solution with DFO-Bn-NCS. Check the chelation efficiency of 89Zr to DFO-Bn-NCS by rad-TLC. Please click here to view a larger version of this figure.
2. Biocompatible formulation of [ 89Zr ]Zr-DBN for cell radiolabeling (Figure 2)
Timing: ~35 min
NOTE: Given the time it takes for the preparation of cells in step 3, start step 3 approximately 20 min before the beginning of step 2 for a ~30 min incubation. This allows cell radiolabeling in step 4 to start within ~5-10 min post-completion of steps 2-3.2.2.
Figure 2: Preparation of the biocompatible formulation of the [89Zr]Zr-DBN for cell radiolabeling. For the preparation of a ready-to-use biocompatible formulation of the radiolabeling synthon, add an equal volume of cell compatible mixture, comprising 1.2 M K2HPO4/KH2PO4 (pH 3.5) + 1.0 M HEPES-KOH to an equal volume of [89Zr]Zr-DBN. Incubate at 25 °C for ~30 min. Please click here to view a larger version of this figure.
3. Preparation of cells for radiolabeling
Timing: ~40-50 min
4. Radiolabeling of cells
Timing: ~125-155 min
Figure 3: Schematic of the initiation of cell radiolabeling. Initiate radiolabeling of cells by the addition of the biocompatibly formulated [89Zr]Zr-DBN to the cell suspension prepared in HEPES buffered Hanks balanced salt solution. Please click here to view a larger version of this figure.
Figure 4: Schematic of cell incubation for radiolabeling. Thoroughly mix the biocompatibly formulated [89Zr]Zr-DBN with the cell suspension and incubate the cell suspension in a temperature-controlled heating block on a shaker for 30-60 min. Please click here to view a larger version of this figure.
Figure 5: Schematic of the quenching of radiolabeling and cell washing. Quench the radiolabeling of cells by the addition of chilled cell medium or H-HBSS, followed by centrifugation at 4 °C. For cell washing, discard the supernatant and resuspend the cell pellet in ~500 µL of chilled cell medium or H-HBSS. Repeat the cycle of discarding the supernatant and resuspending the cell pellet in fresh medium to remove any unbound radiolabeling synthon. Please click here to view a larger version of this figure.
The representative results presented in this manuscript were compiled from the previous [89Zr]Zr-DBN synthesis and cell radiolabeling studies18,19,22,23,24,25. In brief, 89Zr can be successfully complexed with DFO-Bn-NCS in ~30-60 min at 25-37 °C using 7.5-15 µg of DFO-Bn-NCS (Table 2). The cell radiolabeling efficiency varies from 20-50% as an uncorrected yield observed in the radiolabeled cell pellet after washing (Table 1).18,19,22,23,24,25(Table 1).
Overall, following radioactivity concentrations were achieved: 0.1 MBq/106 cells for hepatocytes22,24, 0.50 ± 0.10 MBq/106 for melanoma cells (mMCs)18, 0.47 ± 0.10 MBq/106 for human mesenchymal stem cells (hMSCs)18, 0.39 ± 0.20 MBq/106 for dendritic cells (mDCs)18, 0.27± 0.30 MBq/106 for white blood cells25, and 0.70 ± 0.20 MBq/106 for cardiopoietic stem cells19 (Table 1) . In contrast, no cell labeling was observed using [89Zr]Zr(HPO4)2 or [89Zr]ZrCl425. As demonstrated previously, radiolabels were found to be stable in mMCs, hMSCs, and mDCs, with only negligible percentage of efflux observed over 7 days post-labeling (Figure 6)18. For cardiopoietic stem cells, no efflux was observed over 2 days post-labeling19.
Table 1: Cell radiolabeling efficiency in different cell types with [89Zr]Zr-DBN. Stem cells, melanoma cells, cardiopoietic stem cells, hepatocytes, dendritic cells, and white blood cells were radiolabeled with [89Zr]Zr-DBN with variable cell radiolabeling efficiency. *The cell radiolabeling efficiency (%) was estimated as described in step 4.3.7. Please click here to download this Table.
Table 2: 89Zr chelation efficiency of DFO-Bn-NCS. The chelation efficiency of DFO-Bn-NCS for 89Zr using [89Zr]Zr(HPO4)2 and [89Zr]ZrCl4 are different, as measured by rad-TLC. *Values are presented as average ± standard deviation. With 50 mM DTPA (pH 7.0) as the rad-TLC solvent, [89Zr]Zr(HPO4)2 shows a Rf of ~0.918 and [89Zr]Zr-DBN shows a Rf of ~0.018. With 100 mM DTPA (pH 7.0) as the rad-TLC solvent, [89Zr]ZrCl4 shows a Rf of ~1.0. [89Zr]Zr(HPO4)2 shows a Rf of ~1.0, and [89Zr]Zr-DBN shows a Rf of ~0.021-0.035. Chelation efficiency (%) = percentage of 89Zr chelated to DFO-Bn-NCS to form [89Zr]Zr-DBN = (radioactivity at Rf of [89Zr]Zr-DBN/(sum of the radioactivities at Rf of [89Zr]Zr-DBN and at Rf of [89Zr]Zr(HPO4)2 or [89Zr]ZrCl4) × 100. Please click here to download this Table.
The effect of radiolabeling on the viability of the radiolabeled cells was assessed in mMCs18, pig hepatocytes22,24, hMSCs18, human cardiopoietic stem cells19, and immune cells, such as mouse dendritic cells (mDCs)18 and white blood cells25, using the trypan blue exclusion viability test. It was observed that radiolabeling had no negative impact on the viability of the cells; <5% of the dead cells were found up to 7 days post-labeling for both [89Zr]Zr-DBN-labeled and unlabeled mMCs and hMSCs18. Radiolabeling also did not impact the viability of pig hepatocytes22,24 when tested within 1 h of radiolabeling or 2 days after radiolabeling for the cultured radiolabeled human cardiopoietic stem cells19.
The radiolabeling of human white blood cells also did not negatively impact the viability of cells as ~90-95% of viable cells were observed in both the unlabeled and labeled cell populations within 1 h post radiolabeling25. The radiolabeling of cardiopoietic stem cells did not affect the morphology, viability, or proliferation capacity of human cardiopoietic stem cells19. Functional studies were performed with cardiopoietic stem cells in a myocardial infarct model19. In the functional studies, the characteristic expression of mesodermal and pro-cardiogenic transcription factors defining the cardiopoietic phenotype remained unaltered in radiolabeled cardiopoietic stem cells19. The radiolabeled cardiopoietic stem cells were able to improve the left ventricular ejection fraction in a chronically infarcted mouse heart in the infarcted heart model19.
Figure 6: Stability of various cells radiolabeled with 89Zr over 7 days. The retention of 89Zr radioactivity by radiolabeled cells was found to be stable in all the cells studied, with a negligible efflux observed over 7 days post-labeling. Values are shown as mean ± standard deviation, n = 3. This figure is from Bansal et al.18. Please click here to view a larger version of this figure.
Supplementary Figure S1: Representative rad-TLC of 89Zr chelation with DFO-Bn-NCS using [89Zr]Zr(HPO4)2 in 100 mM DTPA (pH 7.0) solution. Rad-TLC was run in 100 mM DTPA (pH 7.0) as the mobile phase; [89Zr]Zr-DBN ;shows a Rf of ~0.021-0.035 and [89Zr]Zr(HPO4)2 shows a Rf of ~1.0. Chelation efficiency (%) = percentage of 89Zr chelated to DFO-Bn-NCS to form [89Zr]Zr-DBN = [radioactivity at Rf of [89Zr]Zr-DBN/(sum of the radioactivities at Rf of [89Zr]Zr-DBN and at Rf of [89Zr]Zr(HPO4)2)] × 100. Please click here to download this File.
Supplementary Figure S2: Representative rad-TLC of 89Zr chelation with DFO-Bn-NCS using [89Zr]ZrCl4 in 100 mM DTPA (pH 7.0) solution. Rad-TLC was run in 100 mM DTPA (pH 7.0) as the mobile phase; [89Zr]Zr-DBN shows a Rf of ~0.021-0.035 and [89Zr]ZrCl4 shows a Rf of ~1.0. Chelation efficiency (%) = percentage of 89Zr chelated to DFO-Bn-NCS to form [89Zr]Zr-DBN = [radioactivity at Rf of [89Zr]Zr-DBN/(sum of the radioactivities at Rf of [89Zr]Zr-DBN and at Rf of [89Zr]ZrCl4)] × 100. Please click here to download this File.
Supplementary Figure S3: Representative rad-TLC of cell radiolabeling reaction from step 4.2.3 in a 20 mM sodium citrate (pH 4.9-5.1):methanol (1:1, V:V) solution. Rad-TLC was run in 20 mM sodium citrate (pH 4.9-5.1):methanol (1:1, V:V) as the mobile phase; the radiolabeled cells show a Rf of ~0.01-0.02 and [89Zr]Zr-DBN and [89Zr]ZrCl4 show a Rf of ~0.73-0.81. The percentage of radioactivity at a Rf of ~0.01-0.02 is calculated by formula: [(radioactivity at Rf = ~0.01-0.02)/(sum of the radioactivities at Rf = ~0.01-0.02 and Rf = ~0.73-0.81)] × 100. Please click here to download this File.
Supplementary Figure S4: Representative rad-TLC of no-cell control reaction from step 4.2.4 in a 20 mM sodium citrate pH 4.9-5.1: methanol (1:1, V:V) solution. With 20 mM sodium citrate (pH 4.9-5.1): methanol (1:1, V:V) as the rad-TLC solvent, rad-TLC was run in 20 mM sodium citrate (pH 4.9-5.1):methanol (1:1, V:V) as the mobile phase; [89Zr]Zr-DBN and [89Zr]ZrCl4 show a Rf of ~0.73-0.81. The percentage of radioactivity at Rf = ~0.01-0.02 is calculated by formula: [(radioactivity at Rf = ~0.01-0.02)/(sum of the radioactivities at Rf = ~0.01-0.02 and Rf = ~0.73-0.81)] × 100. Please click here to download this File.
Following are critical steps in the protocol that need optimization for effective cell radiolabeling. In protocol steps 1.2 and 1.3, depending on the volume of [89Zr]Zr(HPO4)2 or [89Zr]ZrCl4 employed, an appropriate volume (microliters) of base must be used; 1.0 M K2CO3 solution must be used for the neutralization of [89Zr]Zr(HPO4)2 and 1.0 M Na2CO3 solution for the neutralization of [89Zr]ZrCl4 to achieve a pH range of 7.5-8.0. In step 2.1, the cell-compatible mixture comprising K2HPO4/KH2PO4 and HEPES must be added into the [89Zr]Zr-DBN formulation, when prepared from [89Zr]ZrCl4, to increase the cell radiolabeling efficiency. It is noted that desferrioxamine-Bn-NCS radiolabeled with [89Zr]ZrCl4 failed to radiolabel stem cells unless phosphate and HEPES buffers were added in the cell labeling mixture, indicating that an appropriate formulation is needed to use [89Zr]ZrCl4 as a starting formulation.
For optimization of the cell labeling efficiency for each cell type (protocol step 4.2), the incubation time must be increased to up to ~60 min, and the incubation temperature increased from 25 °C to 37 °C or the pH of the labeling solution increased to ~8.0. The choice of incubation time, temperature, and pH are as per the compatibility of the cell type. For example, radiolabeling of hepatocytes was performed at 27 °C for 45 min at pH ~7.522,24, but radiolabeling of stem cells was performed at 37 °C for 30 min at pH ~7.518.
In protocol step 4.3, to prevent the loss of cells during washing, the supernatant must be pipetted out carefully with a pipette volume setting lower than the supernatant volume that needs to be pipetted out. For example, if the supernatant volume is ~600 µL, then the supernatant must be pipetted out in steps of ~200-500 µL. This helps to avoid disturbing the cell pellet. If the cell pellet gets disturbed while pipetting out supernatant, then pipetting out of the supernatant should be stopped and centrifuged again to get a well-defined cell pellet. Some cells, such as monocytes in the white blood cell suspension are prone to binding to the walls of the tube. This impacts the recovery of all radiolabeled white blood cells from the tube after washing.
After radiolabeling of cells, a visual inspection test, a trypan blue exclusion viability test, and a stability/efflux analysis are acceptable as the quality control criteria. If cell viability is consistently decreasing at every radiolabeling attempt for a particular cell type, then the cells that are being radiolabeled should be checked to ensure they are compatible with the cell medium and buffer used during cell radiolabeling. Various compatible buffers and cell medium are summarized in Table 1. It is preferred to use known compatible cell culture conditions for stability/efflux studies. For example, stability/efflux studies can be performed with stem cells, dendritic cells, macrophages, cancer cells, decidual stromal cells, and peripheral blood mononuclear cells in cell culture conditions as shown by Bansal et al.18and Friberger et al.20.
The potential problems of low chelation efficiency or inconsistent chelation efficiency, and/or low cell radiolabeling yield or loss in cell viability can be resolved by modifying steps 1.5, 4.3.7, and 4.3.10, based on the reasons summarized in Table 3.
Table 3: Troubleshooting related to [89Zr]Zr-DBN synthesis and cell radiolabeling. Possible reasons and solutions for a low/inconsistent chelation efficiency, low cell radiolabeling yield, or loss in cell viability. Please click here to download this Table.
The PET imaging of cell trafficking offers high sensitivity, suitable spatial resolution, and low background25 as advantages over other modalities3,4,9,10. The [89Zr]Zr-DBN-mediated labeling of cell surface proteins with a stable covalent bond offers stability of the radiolabel tag18,19,20,21,22,23,24,25. As a result, [89Zr]Zr-DBN-based cell labeling enhances the reliability and confidence in the radiosignal (positron emission and gammas produced post-annihilation) obtained from the radiolabeled cells. Meanwhile, other methods, such as [89Zr]Zr-oxine-based radiolabeling, are known to encounter efflux of the labeling radiotag over time due to the non-covalent nature of their radiolabeling, producing lower and off-labeled radiosignals27,28.
A limitation of this cell radiolabeling technique is that the radioactivity signals in the PET images are acquired after in vivo administration of live radiolabeled cells but provides no clue if the signal is originating from live radiolabeled cells or from the debris of radiolabeled cells that could have died in the body after the administration.
PET imaging for cell trafficking has enormous potential, as it can support the development of novel cell-based therapies29,30. Physicians and scientists working in cell-based therapies to treat cancers6,7,8,9,10, myocardial infarction3,4,5, myocardial failure1,2, retinal2 and macular2 degeneration, and diabetes2 will find this 89Zr-based cell labeling protocol instrumental, to better understand the responses of their cell-based therapies such as stem cell therapies. Additionally, 89Zr-labeled stem cells31, white blood cells32, dendritic cells33,34, and CAR T-cells35 have great potential to enter the clinical realm within next 3-5 years. Furthermore, pharmaceutical companies may use this technique to develop and validate their cell-based therapeutic agents.
The authors have nothing to disclose.
This work was supported by NIH 5R21HL127389-02, NIH 4T32HL007111-39, NIH R01HL134664, and DOE DE-SC0008947 grants, International Atomic Energy Agency, Vienna, Mayo Clinic Division of Nuclear Medicine, Department of Radiology, and Mayo Clinic Center for Regenerative Medicine, Rochester, MN. All figures were created using BioRender.com.
Acetonitrile | Thermo Fisher Scientific, Inc., Waltham, MA, USA | A996-4 | |
Alpha Minimum Essential Medium | Thermo Fisher Scientific, Inc., Waltham, MA, USA | 12571063 | |
Anion exchange column | Macherey-Nagel, Inc., Düren, Germany | 731876 | Chromafix 30-PS-HCO3 SPE 45 mg cartridge |
Conical centrifuge tubes (15 mL) | Corning Inc., Glendale, AZ, USA | 352096 | Falcon 15 mL high-clarity polypropylene (PP) conical centrifuge tubes |
Dendritic cells | The American Type Culture Collection, Manassas, VA, USA | CRL-11904 | |
DFO-Bn-NCS | Macrocyclics, Inc., Plano, TX, USA | B-705 | p-SCN-Bn-Deferoxamine |
DMSO | Sigma-Aldrich, Inc., St. Louis, MO | 276855 | |
Dose calibrator | Mirion Technologies (Capintec), Inc., Florham Park, NJ, USA | 5130-3234 | CRC -55tR Dose Calibrator |
Dulbecco’s modified Eagle’s medium | The American Type Culture Collection, Manassas, VA, USA | 30-2002 | |
Fetal Bovine Serum (FBS) | The American Type Culture Collection, Manassas, VA, USA | 30-2020 | |
Hanks Balanced Salt solution (HBSS) | Thermo Fisher Scientific, Inc., Waltham, MA, USA | 14025092 | For preparation of H-HBSS |
Hydrochloric Acid (trace metal basis grade) | Thermo Fisher Scientific, Inc., Waltham, MA, USA | A508P212 | |
Melanoma cells | The American Type Culture Collection, Manassas, VA, USA | CRL-6475 | |
Methanol | Sigma-Aldrich, Inc., St. Louis, MO | 34860 | |
Microcentrifuge tube | Eppendorf, Hamburg, Germany | 30108442 | Protein LoBind microcentrifuge tube |
Murine GM-CSF | R&D Systems, Inc., Minneapolis, MN USA | 415-ML-010 | |
Penicillin/Streptomycin | Thermo Fisher Scientific, Inc., Waltham, MA, USA | 15140-122 | |
Phosphate Buffered Saline without Ca2+ and Mg2+ | Thermo Fisher Scientific, Inc., Waltham, MA, USA | 10010023 | For washing cells |
Saline | Covidien LLC, Mansfield, MA, USA | 1020 | 0.9% Sterile Saline Solution |
Shaker | Eppendorf, Hamburg, Germany | T1317 | Thermomixer |
Silica gel-rad-TLC paper sheet | Agilent Technologies Inc., Santa Clara, CA, USA | SGI0001 | iTLC-SG |