KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
|
| ||
| Antibodies | ||
|
| ||
| Anti-c-Rel Antibody (B-6) Alexa Fluor® 488 | Santa Cruz | sc-6955 AF488; RRID:AB_670194 |
| APC anti-mouse CD117 (c-Kit) | Biolegend | Cat#105812, Clone 2B8; RRID:AB_313221 |
| PE/Cyanine7 anti-mouse Ly-6A/E (Sca-1) | Biolegend | Cat#122513, Clone E13-161.7; RRID:AB_756198 |
| APC/Cyanine7 anti-mouse CD16/32 | Biolegend | Cat#156611, Clone S17011E; RRID:AB_2800709 |
| PerCP/Cyanine5.5 anti-mouse CD34 | Biolegend | Cat#128607, Clone HM34; RRID:AB_1279222 |
| APC anti-mouse CD19 | Biolegend | Cat#152409, Clone 1D3/CD19; RRID:AB_2629838 |
| PE/Cyanine7 anti-mouse/human CD45R/B220 | Biolegend | Cat#103221, Clone RA3-6B2; RRID:AB_313004 |
| Pacific Blue™ anti-mouse CD3 | Biolegend | Cat#100214, Clone 17A2; RRID:AB_493645 |
| PerCP/Cyanine5.5 anti-mouse CD49b | Biolegend | Cat#108915, Clone DX5; RRID:AB_1595599 |
| Pacific Blue™ anti-mouse Lineage Cocktail | Biolegend | Cat#133306, Clone 17A2, RB6-8C5, RA3-6B2, Ter-119, M1/70; RRID:AB_11126978 |
| BV711 Rat Anti-Mouse CD44 | BD Biosciences | Cat#563971, Clone IM7; RRID:AB_2738518 |
| APC-R700 Rat Anti-Mouse CD25 | BD Biosciences | Cat#565135, Clone PC61 |
| APC Rat Anti-Mouse CD4 | BD Biosciences | Cat#561091, Clone RM4-5; RRID:AB_10562037 |
| PerCP/Cyanine5.5 anti-mouse CD8a | Thermo Fisher Scientific | Cat#45-0081-82, Clone 53-6.7; RRID:AB_1107004 |
| Ki-67 rat monoclonal antibody (SolA15), eBioscience | ThermoFisher Scientific | Cat#14-5698-82; RRID:AB_10854564 |
| P2RY12 recombinant rabbit monoclonal antibody (4H5L19) | ThermoFisher Scientific | Cat#702516; RRID:AB_2689476 |
| Goat anti-rat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 | ThermoFisher Scientific | Cat#A-11006; RRID:AB_141373 |
| Goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 | ThermoFisher Scientific | Cat#A-11037; RRID:AB_2534095 |
| Anti-NF-kB p65 antibody | Santa Cruz Biotechnology | Cat#sc-372; RRID:AB_632037 |
| Anti-I kappa B alpha antibody | Santa Cruz Biotechnology | Cat#sc-371; RRID:AB_2235952 |
| Anti-Rho-GDI antibody | Sigma-Aldrich | Cat#R3025; RRID:AB_261317 |
| HRP-conjugated Donkey Anti-Rabbit IgG | GE Healthcare | Cat#NA934V; RRID:AB_772206 |
|
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| Biological samples | ||
|
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| Primary ear fibroblast G/R cells | ||
| Primary bone marrow-derived macrophage (BMDM) G/R cells | ||
| Primary microglia G/R cells | ||
| Primary cells from Thymus, Spleen, Lymph nodes and Bone marrow | ||
|
| ||
| Chemicals, peptides, and recombinant proteins | ||
|
| ||
| MEGAshortscript™ T7 Transcription Kit | Thermo Fisher Scientific | AM1354 |
| MEGAclear™ Transcription Clean-Up Kit | Thermo Fisher Scientific | AM1908 |
| CleanCap® Cas9 mRNA | TriLink Biotechnologies | L-7606 |
| Lipopolysaccharide from Salmonella | Enzo | ALX-581-008-L001 |
| rmTNF-a (aa 80-235) | R & D Systems | 410-MT-010 |
| SPY 650 DNA | Cytoskeleton Inc | CY-SC 501 |
| Mouse adult brain dissociation kit | Miltenyi Biotec | Cat#130-107-677 |
| Percoll | GE Healthcare | Cat#17-0891-01 |
| PBS | Gibco | Cat#10010023 |
| DMEM/F-12 media | Gibco | Cat#21041025 |
| Recombinant Mouse M-CSF Protein | R&D Systems | Cat#416-ML-010 |
| Recombinant Human TGF-beta 1 Protein | R&D Systems | Cat#240-B-010 |
| Paraformaldehyde | Sigma-Aldrich | Cat#158127 |
| Goat serum | Sigma-Aldrich | Cat#G9023 |
| Triton X-100 | Sigma-Aldrich | Cat#T8787 |
| BSA | Sigma-Aldrich | Cat#A8806 |
| Sodium azide | Acros organics | Cat#19038-1000 |
| Prolong gold antifade mountant with DAPI | ThermoFisher Scientific | Cat#P36931 |
| Cycloheximide | Sigma-Aldrich | Cat#01810 |
| RIPA cell lysis buffer | Millipore | Cat#20-188 |
| NuPAGE 4–12% Bis-Tris Gel | Invitrogen | Cat#NP0321 |
| NuPAGE MOPS SDS running buffer | Life technologies | Cat#NP0001 |
|
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| Experimental models: Cell lines | ||
|
| ||
| Immortalized ear fibroblast G/R cells | This paper | |
|
| ||
| Experimental models: Organisms/strains | ||
|
| ||
| C57BL/6Ncrl mice | Charles River Laboratories | RRID:IMSR_CRL:027 |
| B6D2F1 mice | Charles River Laboratories | RRID:IMSR_CRL:099 |
| Double knock-in G/R mice | This paper | |
|
| ||
| Recombinant DNA | ||
|
| ||
| mEGFP-RelA dsDNA donor plasmid | Genewiz | Custom made: https://www.genewiz.com/ |
| mScarlet-c-Rel dsDNA donor plasmid | Genewiz | Custom made: https://www.genewiz.com/ |
|
| ||
| Software and algorithms | ||
|
| ||
| MATLAB | MathWorks | |
| ImageJ (Fiji) | National Institute of Health | |
| Imaging data quantification and analysis code | This paper | https://github.com/Toufiq54/LiveCell_NFkB.git |
| FlowJo Ver.10 | BD Biosciences | https://www.flowjo.com |
| ANY-Maze Ver. 7.1 | Stoelting | Cat#60000 |
| Video Freeze Ver. 3.01 | MED-Associates | Cat#SOF-843 |
|
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| Other | ||
|
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| Cell strainers | Fisher Scientific | Cat#22-363-548 |
| Grip strength meter | Bioseb | Cat#BIO-GS3 |
| Mouse fear conditioning chamber | MED-Associates | Cat#MED-VFC-USB-M |