FR positive cancer cell lines (e.g., KB or MDA-MB-231 cells) were seeded at density of 104 cells/100 μl media into 96 well plates and grown overnight. Anti-fluorescein CAR T cells were added to each well in the absence or presence of bispecific adapters. After co-incubation for 6–24 h, plates were centrifuged at 350xg for 10 min to remove debris and supernatants were analyzed for lactate dehydrogenase release (cell death analysis) using PierceTM LDH cytotoxicity assay kit (Thermo Fisher Scientific, MA) and interferon γ (IFNγ) levels using human IFNγ ELISA kit (Biolegend, CA), while pellets were either evaluated for CAR T cell activation by staining with anti-human CD69 APC (1:100 dilution, Biolegend, catalog#: 310910) or examined for CAR T cell proliferation by culturing for 5 days in TexMACSTM medium (Miltenyi Biotech Inc, CA) containing 1% penicillin and streptomycin sulfate and quantitating by flow cytometry using the intrinsic GFP fluorescence and staining with anti-human CD3 APC antibody (1:100 dilution, Biolegend, catalog#: 300312).
Texmacs medium
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
TexMACS medium is a cell culture medium developed by Miltenyi Biotec for the in vitro expansion of T cells. It is designed to support the proliferation and maintenance of T cells while preserving their functionality.
Automatically generated - may contain errors
Lab products found in correlation
T cell transact, by Miltenyi Biotec (13 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (8 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (6 mentions)
Interleukin 7 (il 7), by Miltenyi Biotec (6 mentions)
Il 15, by Miltenyi Biotec (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Human il 7, by Miltenyi Biotec (5 mentions)
Human ab serum, by Merck Group (5 mentions)
Human il 2, by Miltenyi Biotec (4 mentions)
Human il 15, by Miltenyi Biotec (4 mentions)
Macs gmp t cell transact, by Miltenyi Biotec (4 mentions)
Clinimacs prodigy, by Miltenyi Biotec (3 mentions)
Histopaque 1077, by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
X vivo 15 medium, by Lonza (3 mentions)
Recombinant human il 2, by Miltenyi Biotec (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Apc anti human cd69, by BioLegend (2 mentions)
Human pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
120 protocols using texmacs medium
1
CAR T Cell Cytotoxicity and Activation Assay
2
Regulatory T-cell Differentiation Protocol
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For regulatory T‐cell (Treg) differentiation, CD4+ T cells were incubated for 5 days using TexMACSTM medium (# 130‐097‐196, Miltenyi Biotec, Bergisch Gladbach, Germany), stimulated with T Cell TransActTM human (Miltenyi Biotec, Bergisch Gladbach, Germany) and supplemented with 5 ng/ml TGFβ1, 1 µg/ml anti‐IFNγ, 1 µg/ml anti‐IL4, 100 U/ml IL2 (#130 095‐067 | #130 095‐743 | # 130‐095‐709 | #130 097 742, Miltenyi Biotec, Bergisch Gladbach, Germany), and 10 mM of retinoic acid ( #R2625; Sigma‐Aldrich, Darmstadt, Germany).
3
Generation of Clinical-Grade CAR T Cells
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Buffy coats from consenting healthy anonymous donors were obtained from the German Red Cross Dortmund as registered and approved by the Ethics Committee of the German Red Cross. All study-related procedures have been performed according to the Declaration of Helsinki and to the relevant ethical guidelines.
CAR T cells were generated according to previously applied protocols 46 (link). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation and subsequent T cells enrichment was performed using the human pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Isolated T cells were cultivated in TexMACSTM Medium (Miltenyi Biotec) supplemented with 200 IU/ml of recombinant human IL-2 IS, research grade (Miltenyi Biotec) and activated with TransActTM, human (Miltenyi Biotec). Twenty-four hours after activation, lentiviral vector was added for transduction. T Cell TransActTM and excess viral vector were removed 3 days post activation and replaced with fresh TexMACSTM Medium, supplemented with 200 IU/mL IL-2, for further cultivation of T cells. Cell splitting and feeding occurred in a 2-3 days interval. After 12 days of expansion, T cells were subjected to experimental studies.
CAR T cells were generated according to previously applied protocols 46 (link). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation and subsequent T cells enrichment was performed using the human pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Isolated T cells were cultivated in TexMACSTM Medium (Miltenyi Biotec) supplemented with 200 IU/ml of recombinant human IL-2 IS, research grade (Miltenyi Biotec) and activated with TransActTM, human (Miltenyi Biotec). Twenty-four hours after activation, lentiviral vector was added for transduction. T Cell TransActTM and excess viral vector were removed 3 days post activation and replaced with fresh TexMACSTM Medium, supplemented with 200 IU/mL IL-2, for further cultivation of T cells. Cell splitting and feeding occurred in a 2-3 days interval. After 12 days of expansion, T cells were subjected to experimental studies.
4
Evaluating EBV Peptide Immunogenicity
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The immunogenic potential of EBV-derived peptides was evaluated by functional immunoassays. PBMCs were isolated from blood of healthy HLA-A*03:01+EBV+ donors by density gradient centrifugation. Freshly isolated PBMCs were rested overnight (at 37°C, 5% CO2) at a density of 1 × 107 cells/well (24-well-plate, Sarstedt) prior to short- (overnight) and long-term (7 days) in vitro stimulation. The known HLA-A*03:01-restricted EBV-derived peptide RLRAEAQVK (A*03_EBNA3ARLRA, ProImmune [17 (link), 47 (link)]) and the peptide pool PepTivator EBV Consensus (EBV_Consensus, Miltenyi Biotec) have consistently been used as referential stimulating antigens.
On day 1, cells at a density of 5 × 106 cells/well (24-well-plate, at 37°C, 5% CO2) were stimulated with one of the eleven EBV-specific peptides (10 μg/ml) or with EBV_Consensus+4PMIX (Table1 ) consisting of a combination of EBV_Consensus (1μg per peptide/ml) and the four highly immunodominant peptides (10 μg per peptide/ml). Cells were expanded in TexMACS-medium (Miltenyi Biotec) supplemented with 0.5 μl/ml interleukin (IL)-2 and 1 μl/ml IL-7 (both PeproTech) over 7 days.
On day 1, cells at a density of 5 × 106 cells/well (24-well-plate, at 37°C, 5% CO2) were stimulated with one of the eleven EBV-specific peptides (10 μg/ml) or with EBV_Consensus+4PMIX (Table
5
Cytokine Profiling of CAR-T Cells
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2.5 × 104 CAR-expressing cells were seeded in 96 well flat-bottom plates. The supernatant of AsPC-1 WT and AsPC-1-TGF was harvested and processed as described previously. 50 µL tumor-derived supernatant was added together with aCD66c and aLAP. The assay was performed in 200 µL TexMACS™ Medium (Miltenyi Biotec, catalog no.: 130-097-196) without cytokines for 24 h. Cytokine concentration was measured by cytokine multiplex analysis (Miltenyi Biotec, catalog no.: 130-099-169). The expression of activation markers was determined by flow cytometric analysis.
6
Isolation and Transduction of Primary T Cells
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For in vitro assays, buffy coats from healthy donors were obtained from DRK Dortmund. Density gradient centrifugation with Pancoll human (PAN-Biotech, catalog no.: P04-601000) was used for PBMC isolation. T cells were isolated from PBMCs with the Pan T Cell Isolation Kit, human (Miltenyi Biotec, catalog no.: 130-096-535). Isolated T cells were cultured in TexMACS™ Medium (Miltenyi Biotec, catalog no.: 130-097-196) supplemented with 12.5 ng/mL IL-7 (Miltenyi Biotec, catalog no.: 170-076-111) and 12.5 ng/mL IL-15 (Miltenyi Biotec, catalog no.: 170-076-114). For activation before transduction, T cells were seeded in a 24-well plate at a density of 1 × 106 cells/mL and T Cell TransAct™ (Miltenyi Biotec, catalog no.: 130-111-160) was added. After 24 h, the cells were transduced with an MOI of 10. After 3 d, the culture medium was exchanged completely to remove residual LVV particles. T cells were cultured for 10–14 d with the regular addition of fresh culture medium every 2 d. CAR T cells for in vivo studies were manufactured using the CliniMACS Prodigy™ (Miltenyi Biotec) as described previously by Lock et al.30 (link) As starting material leukapheresis obtained from “Institut für klinische Transfusionsmedizin und Immungenetik Ulm” was used. The enriched T cells were transduced at an MOI of 10 and cultured until day 10 for harvesting.
7
Evaluating CAR T Cell Efficacy Against Pancreatic Cancer
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1 × 104 AsPC-1 WT or 1 × 104 AsPC-1-TGF were seeded in 96-well flat-bottom plates. After 24 h 2.5 × 104 CAR-expressing cells, as well as aCD66c and aLAP, were added. The assay was performed in 200 µL TexMACS™ Medium (Miltenyi Biotec, catalog no.: 130-097-196) without cytokines. Tumor growth was monitored by live-cell imaging (IncuCyte® S3, Sartorius). The area per well covered by GFP+ target cells was used as a metric to measure AdCAR T cell efficacy. All values were normalized to the starting point.
8
Human PBMC Isolation and Immune Cell Culture
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Human peripheral blood mononuclear cells (PBMC) were isolated from the blood using Ficoll Paque Plus (Sigma-Aldrich). The subtypes of MHC I molecules of Human PBMC were examined and HLA-A2+ human PBMC were identified by flow cytometry. Primary T cells were activated and expanded by culturing 1 × 106 PBMC in TexMACS medium (Miltenyi Biotec, Germany) supplemented with 500 IU/ml human recombinant Interleukin 2 (rhIL2) (Miltenyi Biotec), with 10 µl TransAct (Miltenyi Biotec) which contains anti-CD3 and CD28 antibodies, for 3 days. The T cells were then purified with a pan-T cell isolation kit (Miltenyi Biotec) and cultured in TexMACS medium + rhIL2 for 2 weeks before CD3/28 antibody re-stimulation or DC cell-based antigen stimulation. Primary NK cells were activated and expanded by culturing 1 × 106 PBMC in NK MACS medium (Miltenyi Biotec) supplemented with 5% human AB serum (Invitrogen) and 500 IU/ml rhIL2, with 5 × 105 microbeads conjugated with anti-CD335 and CD2 antibodies (Miltenyi Biotec) for 7 days. The NK cells were purified with NK cell isolation kit (Miltenyi Biotec) and cultured in NK MACS medium + human AB serum + rhIL2 for 1 week before hypoxic culture. The purity of the immune cells was examined by flow cytometry analysis.
9
Isolation and Activation of UniCAR T-cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (German Red Cross, Dresden) using density gradient centrifugation with Pancoll separation solution (PanBiotech). T-cells were separated from the PBMCs via magnetic isolation using Pan T-cell Isolation Kit from Miltenyi Biotec according to the manufacturer’s instructions. After isolation, T-cells were incubated in RPMI complete medium supplemented with IL-2. Isolated T-cells were furthermore activated using T-Cell TransAct™ (Miltenyi Biotec) and lentiviral transduced with the UniCAR construct as previously described [26 ]. Subsequent culture and expansion were performed in TexMACS™ medium (Miltenyi Biotec) supplemented with human IL-2, IL-7 and IL-15 (Miltenyi Biotec). UniCAR T-cells were cultured in RPMI medium without cytokines for 24 h prior to experiments.
10
Baboon PBMC Activation Assay
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Baboon PBMCs extracted at different time-points were cultured in 96-well plates at 2 × 106 cells/mL in TexMACS medium (Miltenyi) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin for 48 h. PBMCs were stimulated with coated anti-CD3 Ab (SP34; 1 µg/mL), human IL-2 (300 UI/mL) or human IL-7 (10 ng/mL; Miltenyi). T lymphocyte activation was measured by CD25 (MA251) and CD69 (FN50) staining, followed by flow cytometry.
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