Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference |
Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Alexa Fluor 647 anti-biotin IgG (mouse monoclonal) | Jackson Immuno Labs | Cat# 200-602-211 RRID:AB_2339046 |
|
| Antibody | Alexa Fluor 488 anti-biotin IgG (mouse monoclonal) |
Jackson Immuno Labs | Cat# 200-542-211 RRID:AB_2339040 |
|
| Sequence-based reagent | Receptor DNA strand | This paper | Benzylguanine-5'- AATATGATGTATGTGG-3' | Oligonucleotide was ordered from IDT with a 5' terminal amine. Conjugation to benzyl-guanine was performed as described (Farlow et al., 2013). |
| Sequence-based reagent | DNA ligand strand | IDT | Biotin-5'-TTTT-TTTCATACATCATATT- 3'-Atto647 | |
| Sequence-based reagent | p8064 DNA scaffold | IDT | Cat# 1081314 | |
| Chemical compound, drug | Alexa Fluor 488 Phalloidin | Thermo/Molecular Probes | Cat# A12379 | |
| Commercial assay, kit | Lipofectamine LTX | ThermoFisher | Cat# 15338030 | |
| Commercial assay, kit | Lenti-X Concentrator | Takara Biosciences | Cat# 631231 | |
| Peptide, recombinant protein | Pierce Biotinylated Bovine Serum Albumin (Biotin-LC-BSA) | ThermoScientific | Cat# 29130 | |
| Peptide, recombinant protein | Neutravidin | ThermoScientific | Cat# 31050 | |
| Cell line (human) | Lenti-X 293 T cell line | Takara Biosciences | Cat# 632180 | For lentivirus production. |
| Cell line (human) | HEK293T cells | UCSF Cell Culture Facility | For lentivirus production. | |
| Cell line (mouse) | Raw264.7 Macrophages | ATCC | Cat# ATCC TIB-71 RRID:CVCL_0493 |
|
| Cell line (human) | THP1 Monocytes | ATCC | Cat# ATCC TIB-202 RRID:CVCL_0006 |
|
| Transfected construct (mouse) | pHR-DNA-CARγ | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB - P20491 (FCERG_MOUSE) linker: GSGS, Fluorophore: mGFP or BFP. | |
| Transfected construct (mouse, human) | pHR-Syk-BFP | Adapted from DOI: 10.1016/j.immuni.2020.07.008 | CDS: aa1-629 UniProtKB - P48025 (KSYK_MOUSE), Linker: ADPVAT, Fluorophore: BFP. | |
| Transfected construct (mouse, human) | pHR-DNA-CARadhesion | DOI: 10.1016/j.immuni.2020.07.008 | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), linker: SADASGG, fluorophore: eGFP. | |
| Transfected construct (mouse) | pHR-DNA-CARγTM-C25A | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: (aa 19–86) of the Fcγ-chain UniProtKB – P20491 (FCERG_MOUSE) with aa25 mutated from C to A linker: GSGS, fluorophore: mGFP or BFP. |
|
| Transfected construct (mouse) | pHR-DNA-CARγTM-aa26-86 | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: (aa 26–86) of the Fcγ-chain UniProtKB – P20491 (FCERG_MOUSE) linker: GSGS, fluorophore: mGFP or BFP. |
|
| Transfected construct (mouse) | pHR-DNA-CARγ-αTM (aa291–404) | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLC LLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: FcGR1 α-chain (aa 291–404) UniProtKB – P26151 (FCGR1_MOUSE) followed by cytoplasmic domain (aa 45–86) of the Fcγ-chain UniProtKB - P20491 (FCERG_MOUSE) linker: GSGS, fluorophore: mGFP or BFP. |
|
| Transfected construct (mouse, human) | pHR-mNeonGreen-tSH2 Syk | Adapted from DOI: 10.1016/j.cell.2018.05.059 | CDS: aa2-261 UniProtKB - P48025 (KSYK_MOUSE), linker: GGGSGGGG, fluorophore: mNeonGreen. | |
| Transfected construct (mouse, human) | pHR-Akt PH domain | This paper | CDS: aa1–164 UniProtKB – P31749 (AKT1_HUMAN), linker: HMTSPVAT, fluorophore: mGFP. | |
| Transfected construct (mouse) | pHR-DNA-CAR4xγ | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), four repeats of the cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE) with a GSGS linker between each repeat, linker: GSGS, fluorophore: mGFP. |
|
| Transfected construct (mouse) | pHR-DNA-CAR-N1xγ−3xΔITAM | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), the cytoplasmic domain (aa 45–86) of the Fcγ-chain UniProtKB – P20491 (FCERG_MOUSE) followed by three repeats of the cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE) with aa65 and aa76 mutated from YtoF and a GSGS linker between each repeat, linker: GSGS, fluorophore: mGFP. |
|
| Transfected construct (mouse) | pHR-DNA-CAR-3xΔITAM-C1xγ | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), three repeats of the cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE) with aa65 and aa76 mutated from YtoF and a GSGS linker between each repeat followed by the cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE), linker: GSGS, fluorophore: mGFP. |
|
| Transfected construct (mouse) | pHR-DNA-CAR-1xΔITAM-1xγ−2xΔITAM | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), the cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE) with aa65 and aa76 mutated from YtoF the cytoplasmic domain, GSGS linker (aa 45–86) of the Fcγ-chain UniProtKB – P20491 (FCERG_MOUSE), GSGS linker, followed by two more repeats of the Fc γ-chain UniProtKB – P20491 (FCERG_MOUSE) with aa65 and aa76 mutated from YtoF the cytoplasmic domain and a GSGS linker between each repeat, linker: GSGS, fluorophore: mGFP. |
|
| Transfected construct (human) | pHR-DNA-CARγ human | This paper | In PhR vector. Signal peptide: (MQSGTHWRVLGLCLLSVGVWGQD) derived from CD3ε Extracellular: HA tag plus a linker (LPETGGGGGG), SNAPf (from the pSNAPf plasmid, New England Biolabs) Linker: GGSGGSGGS, TM and intracellular: CD86TM (aa 236–271), cytoplasmic domain (aa 45–86) of the Fc γ-chain UniProtKB – P30273 (FCERG_HUMAN) linker: GSGS, fluorophore: mGFP or BFP. |
|
| Recombinant DNA reagent | pMD2.G lentiviral plasmid | D. Stainier, Max Planck; VSV-G envelope | RRID:addgene_12259 | |
| Recombinant DNA reagent | pCMV-dR8.91 | DOI: 10.1038/nature11220. | Current RRID:addgene_8455 | |
| Recombinant DNA reagent | pHRSIN-CSGW | DOI: 10.1038/nature11220. | ||
| Software, algorithm | ImageJ | NIH | ||
| Software, algorithm | Affinity Designer | |||
| Software, algorithm | Fiji | https://fiji.sc/ | ||
| Software, algorithm | Prism | GraphPad | 8 | |
| Software, algorithm | Micromanager | DOI:10.14440/jbm.2014.36 | ||
| Other | 5 μm silica microspheres | Bangs | Cat# SS05N | |
| Other | Biotinyl Cap PE | Avanti | Cat# 870273 | |
| Other | POPC | Avanti | Cat# 850457 | |
| Other | PEG5000-PE | Avanti | Cat# 880230 | |
| Other | Atto390 DOPE | ATTO-TEC GmbH | Cat# AD 390-161 | |
| Other | MatriPlate | Brooks | Cat# MGB096-1-2-LG-L | |
| Other | 96-well round bottomed plates | Corning | Cat# 38018 | |
| Other | Illustra NAP-5 columns | Cytiva | Cat# 17085301 |