Spleens were passed through 40μm cells strainers to generate single cell suspensions and stained for with the relevant antibodies. For BATF and IRF4 staining, cells were fixed (30 minutes) and permeabilized with the Foxp3/Transcription Factor Staining buffer (ThermoFisher Scientific) prior to intracellular staining. For Y-ae staining, splenocytes were cultured ex vivo in DMEM/10% FBS/Hepes/Pen/Step with HEL-EaGFP or HEL-BSA at 3ug/mL for 2 hrs at 37°C prior to antibody staining. For BrdU stains, cells were fixed/permeabilized with BD BrdU flow kit following manufacturer’s instructions, and stained using the 3D4 antibody. EdU staining was performed on samples fixed for 30 mins with BD Cytofix/Cytoperm, following manufacturer’s instructions (Click-iT™ Plus kit, ThermoFisher Scientific). All permeabilization steps included a short vortex pulse and were performed with an overnight incubation at 4°C. Data acquisition was carried out using BD LSR II, LSR Fortessa, LSR Fortessa X20, FACSymphony cytometers. FACs sorting was performed using the FACs Aria II SORP or Fusion II machines with 85um or 100um nozzles. Analysis was performed with Flowjo (Treestar Inc.).
Click it plus kit
Manufactured by Thermo Fisher Scientific
The Click-iT™ Plus kit is a tool for detecting and quantifying newly synthesized proteins in cells. It utilizes a chemical reaction to label newly synthesized proteins, enabling their visualization and analysis.
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Lab products found in correlation
Lsrfortessa, by BD (4 mentions)
Brdu flow kit, by BD (2 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Celltiter glo reagent, by Promega (2 mentions)
Sp8 microscope, by Leica (1 mentions)
Donor calf serum, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions)
Miniprep kit, by Promega (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Fitc annexin 5 kit, by BioLegend (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Cayman Chemical (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Upright fluorescent microscope, by Zeiss (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
15 protocols using click it plus kit
1
Multiparametric Analysis of Immune Cells
2
3D Imaging of Proliferating Mammary Glands
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Mice were injected intraperitoneally with 100 mg/kg of ethynyldeoxyuridine (EdU) to label proliferating cells. Animals were deeply anesthetized using isoflurane and perfused with PBS containing heparin followed by tissue fixation using 4% paraformaldehyde (PFA), as described previously.65 The tissue was harvested and subjected to immunolabeling using a previously published staining protocol that avoided methanol treatment steps.66 Antibodies to smooth muscle actin (SMA) and keratin‐8 (KRT8) were used. EdU+ TEBs were identified by AF647 labeling using the Click‐IT Plus kit (Thermo Fisher Scientific). Glands were cleared using the FluoClearBABB method,67 and high‐resolution 3‐dimensional images acquired using a Leica SP8 microscope equipped with a white light laser and Leica BABB immersion lenses (HCX PL FLUOTAR 5×/0.15 IMM lens for low‐resolution and HCX APO L 20×/0.95 IMM lens for high resolution images). Imaging data was analyzed on a power workstation using Imaris software (Bitplane).
3
Quantifying Neuronal Differentiation and Apoptosis
4
Isolation and Transduction of Rat Tanycytes
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Tanycytes were isolated from 10 days old rat as previously described69 (link) and cultured in DMEM/F12 (31966, Thermo Fisher Scientific) supplemented with 10% (v/v) donor calf serum (Invitrogen), 1% (v/v) L-glutamine (Thermo Fisher Scientific), and 2% penicillin/streptomycin. The tanycytes were plated on inserts (Grenier bio-one 665641) at a density of 442 cells/mm2 and were transduced the same day with the PKCαWT (MOI 10) and PKCαD463H (MOI 20) lentiviruses in the presence of 1 μg/ml of polybrene. The following day the medium was changed. Two days post transduction EdU (1.6 μg/ml) was added to the medium; 12 h later (day 3 post transduction) the cells were fixed and processed for immunostaining as described for astrocytes. EdU staining was chemically revealed with the Click-iT plus kit (C10640, Thermo Fisher Scientific).
5
Detecting S-phase Cells with EdU
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To detect cells undergoing S-phase, 5-ethynyl-2′-deoxyuridine (EdU; 10 mg/ml, Thermo Fisher Scientific) was injected into the lateral ventricle in developing chick embryos (0.1 µl) and the peritoneum in pregnant mice (100 mg/kg) 1 h before fixation. EdU detection was with the Click-iT Plus Kit (Thermo Fisher Scientific).
6
Multiparametric Analysis of Immune Cells
7
Clonogenic, UV, and Chemosensitivity Assays
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For clonogenic experiments, 1000–2000 cells (depending on plating efficiency) were seeded in six-well plates. For UV sensitivity, cells were treated 24 h after seeding. For cisplatin and olaparib treatment, cells were seeded in indicated drug concentrations for 24 and 72 h, respectively, followed by media change. Two weeks later, colonies were stained with Crystal violet. For time-course proliferation experiments, 500 cells were seeded in wells of 96-well plates, and cellular viability was scored at indicated days using the CellTiterGlo reagent (Promega G7572). EdU incorporation was assayed using the Click-iT Plus kit (Invitrogen C10633 ) according to the manufacturer’s instructions, using a BD FACSCanto II flow cytometer, and analyzed using the FACSDiva 8.0.1 software.
For the SupF assay36 (link), cells were transfected with UVC-irradiated (1000 J/m2) pSP189 (SupF) plasmid. Three days later, the plasmid was recovered using a miniprep kit (Promega), DpnI digested and transformed into MBM7070 indicator bacteria. Transformants were selected on plates containing 1 mM IPTG and 100 μg/ml X-gal. The ratio of white (mutant) to total (blue + white) colonies was scored as mutation frequency.
For the SupF assay36 (link), cells were transfected with UVC-irradiated (1000 J/m2) pSP189 (SupF) plasmid. Three days later, the plasmid was recovered using a miniprep kit (Promega), DpnI digested and transformed into MBM7070 indicator bacteria. Transformants were selected on plates containing 1 mM IPTG and 100 μg/ml X-gal. The ratio of white (mutant) to total (blue + white) colonies was scored as mutation frequency.
8
Comprehensive Cell Proliferation and Survival Assays
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Apoptosis was quantified using the FITC Annexin V kit (Biolegend 640906). For cell-cycle profiles, cells were fixed in 4% paraformaldehyde and stained with FxCycle PI reagent (Invitrogen F10797). EdU incorporation was assayed using the Click-iT Plus kit (Invitrogen C10633). For clonogenic experiments, 250 cells were seeded in 6-well plates and, 2 weeks later, stained with Crystal violet or trypsinized and counted using an automated cell counter for quantification of cellular proliferation. When HU sensitivity was analyzed, cells were incubated with 0.2 mM HU immediately after seeding; 72 h later, media was replaced and plates were stained with Crystal violet 2 weeks later. For time-course proliferation experiments, 500 cells were seeded in wells of 96-well plates and cellular viability was scored at indicated days using the CellTiterGlo reagent (Promega G7572).
9
Evaluating EpCAM CAR-T Cell Efficacy
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EpCAM CAR-modified or untransduced control thy1.1 + T cells (5 × 106) were 1:1 mixed with thy1.2+ untransduced control T cells (5 × 106), after which mixed T cells were intravenously injected into thy1.1+ thy1.2+ B6 mice. Three days later, peripheral blood was subjected to flow cytometry analysis to evaluate proportions of transferred T cells.
EGFRvIII-specific CAR-T cells containing the same intracellular signaling domains as EpCAM CAR-T were used as a control. 1 × 107 thy1.2+ derived EGFRvIII CAR-T cells or EpCAM CAR-T cells were transferred into thy1.1+ B6 mice separately. Mice were euthanized on day 3 post T cell infusion, and spleen, colon, and lung were dissected, minced, and digested to obtain a single-cell suspension. All samples were incubated in ACK lysis buffer on ice for 30 sec. Numbers of transferred and recipient T cells in each mouse were analyzed by flow cytometry. To measure in vivo proliferation, 12 h before flow analysis, mice were intraperitoneally injected with 20 mg/kg 5-ethynyl-2´-deoxyuridine (EdU) (Cayman Chemical). Transferred CAR-T cell proliferation (indicated by EdU staining) was analyzed using a Click-iT Plus kit (Invitrogen™) according to the manufacturer’s instructions.
EGFRvIII-specific CAR-T cells containing the same intracellular signaling domains as EpCAM CAR-T were used as a control. 1 × 107 thy1.2+ derived EGFRvIII CAR-T cells or EpCAM CAR-T cells were transferred into thy1.1+ B6 mice separately. Mice were euthanized on day 3 post T cell infusion, and spleen, colon, and lung were dissected, minced, and digested to obtain a single-cell suspension. All samples were incubated in ACK lysis buffer on ice for 30 sec. Numbers of transferred and recipient T cells in each mouse were analyzed by flow cytometry. To measure in vivo proliferation, 12 h before flow analysis, mice were intraperitoneally injected with 20 mg/kg 5-ethynyl-2´-deoxyuridine (EdU) (Cayman Chemical). Transferred CAR-T cell proliferation (indicated by EdU staining) was analyzed using a Click-iT Plus kit (Invitrogen™) according to the manufacturer’s instructions.
10
Visualizing Proliferation and Apoptosis
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