Antibodies |
|
|
|
Anti-TLR2 |
Abcam |
Cat# ab1655; RRID: AB_302428 |
Anti-TLR2 neutralizing |
InvivoGen |
Clone T2.5; Cat# mab-mtlr2; RRID: AB_763722 |
Mouse Control IgG1 |
InvivoGen |
Cat# mabg1-ctrlm; RRID: AB_11203233 |
Anti-C3 |
MP-Biomedicals |
Cat# 855444 |
Anti-C3 |
Abcam |
Cat# ab11887; RRID: AB_298669 |
Anti-C3d |
Agilent |
Cat# A006302; RRID: AB_578478 |
Anti-CFB |
Atlas Antibodies; Sigma |
Cat# HPA001817; RRID: AB_1078779 |
Anti-CFD |
R&D systems |
Cat# MAB5430; RRID: AB_10640506 |
Rat IgG1 Isotype Control |
R&D systems |
Cat# MAB005; RRID: AB_357348 |
MAC/ anti-C5b-9 for Mouse IHC |
Biozol |
Cat# FGI-10–1801 |
MAC/ anti-C5b-9 for human IHC |
Santa Cruz Biotechnology |
clone aE11; Cat# sc58935; RRID: AB_1119839 |
MAC/ anti-C5b-9 for WB |
Santa Cruz Biotechnology |
Clone 2A1; Cat# sc66190; RRID: AB_1119840 |
Anti-CEP |
Dr. Rowan (Rowan et al., 2017) |
NA |
ZO-1 |
Thermo Fisher Scientific |
Cat# 40–2200; RRID: AB_2533456 |
IBA1 |
Wako |
Cat# 019–19741; RRID: AB_839504 |
Biotinylated Goat Anti-Rabbit IgG |
Vector Laboratories |
Cat# BA-1000; RRID: AB_2313606 |
Biotinylated Rabbit Anti-Goat IgG |
Vector Laboratories |
Cat# BA-5000; RRID: AB_2336126 |
Anti-human CD16 |
BioLegend |
Clone 3G8; Cat# 302006; RRID: AB_314206 |
Anti-human CD86 |
Miltenyi Biotec |
Clone FM95; Cat# 130–094-877; RRID: AB_10839702 |
Anti-human CD45 |
BioLegend |
Clone 2D1; Cat# 368506; RRID: AB_2566358 |
Anti-CD66b |
BioLegend |
Clone G10F5; Cat# 305116; RRID: AB_2566605 |
Anti-human CD14 |
Miltenyi |
Clone Tük4; Cat# 130–098-058; RRID: AB_2660173 |
Anti-human CD80 |
Miltenyi |
Clone 2D10; Cat# 130–099-710; RRID: AB_2659260 |
Alexa Fluor 488 Phalloidin |
Thermo Fisher Scientific |
Cat# A12379; RRID: AB_2315147 |
Alexa Fluor 647 Donkey Anti-Mouse |
Abcam |
Cat# AB150107
|
Alexa Fluor 488 Goat Anti-Rabbit |
Thermo Fisher Scientific |
Cat #A11034; RRID: AB_2576217 |
Alexa Fluor 488 Goat anti-Mouse |
Thermo Fisher Scientific |
Cat #A11001; RRID: AB_2534069 |
|
Biological Samples |
|
|
|
Human donor eye tissue |
The Iowa Lions Eye Bank |
https://iowalionseyebank.org/ |
Peripheral Blood Mononuclear Cells (PBMCs) |
Isolated from healthy volunteers |
NA |
Primary Microglia |
Isolated as per Fernando et al., 2016
|
NA |
Normal Human Serum |
Sigma |
Cat# H4522–20ml |
|
Chemicals, Peptides, and Recombinant Proteins |
|
|
|
Mouse M-CSF |
Miltenyl Biotec |
Cat# 130–094-129 |
Mouse Recombinant GM-CSF |
Stem Cell Technologies |
Cat# 78017.1 |
Pam3Cys4 |
Invivogen |
Cat# tlrl-pms |
CEP-HSA |
Prof. Salomon (Gu et al., 2003) |
|
Mal (TIRAP) Inhibitor Peptide |
Calbiochem |
Cat# 613570–1MG |
Sodium Iodate |
Sigma |
Cat # S4007–100G |
Hoechst |
Sigma |
Cat #B2261–25MG |
Collagen IV |
Sigma |
Cat# C5533–5M |
|
Critical Commercial Assays |
|
|
|
In SITU Cell Death Detection Kit, TMR red |
Roche |
Cat# 12156792910 |
MCP-1 ELISA |
Tebubio |
Cat# 900-K31 |
MAC ELISA |
Abbexa |
Cat# abx350654 |
Isolate II RNA Extraction Kit |
Bioline |
Cat# BIO-52073 |
MMLV Reverse Transcriptase |
Promega |
Cat# M1705 |
SensiFast SYBER Green |
Bioline |
Cat# BIO-92020 |
Pierce LDH Viability Assay |
Thermo Fisher Scientific |
Cat# 13464269 |
LIVE/DEAD Aqua |
Thermo Fisher Scientific |
Cat# L34957
|
Mouse Vectastain ABC HRP Kit |
Vector Laboratories |
Cat#PK-4002; RRID: AB_2336811 |
ImmPACT DAB Substrate |
Vector Laboratories |
Cat#SK-4105; RRID: AB_2336520 |
Vectastain Elite ABC HRP kit |
Vector Laboratories |
Cat#PK-6200; RRID: AB_2336826 |
Vector VIP Peroxidase (HRP) Substrate Kit |
Vector Laboratories |
Cat# SK-4600; RRID: AB_2336848 |
|
Experimental Models: Cell Lines |
|
|
|
ARPE19 |
ATCC |
CRL-2302 |
Immortalized BMDMS |
Prof. Golenbock, UMASS, USA |
N/A |
hfRPE |
Dr. A. Maminishkis (Maminishkis et al., 2006) |
N/A |
THP-1 |
ATCC |
TIB-202 |
HEK293-TLR2 |
Dr. K. Fitzgerald, UMASS, USA |
N/A |
|
Experimental Models: Organisms/Strains |
|
|
|
C57Bl6 |
Jackson labs |
|
Tlr2–/– |
Jackson labs |
JAX stock #004650 |
DBA/2J |
Jackson labs |
JAX stock #000671 |
|
Oligonucleotides |
|
|
|
Primers for SYBER qPCR, |
This paper |
N/A |
|
Recombinant DNA |
|
|
|
C3 luciferase (T1 del) |
|
Addgene |
|
Software and Algorithms |
|
|
|
GraphPad Prism |
GraphPad Software |
https://www.graphpad.com |
ImageJ |
|
https://imagej.nih.gov/ij/ |
FlowJo |
Tree star |
https://www.flowjo.com |