Nunc lab tek permanox chamber slides

The REEP6 splice variant open reading frames were amplified from control D124 optic cup cDNA¹⁶ (link) using the primers REEP6_F1, 5′-GCTAGCCACCATGGACGGCCTGAGGCAGCGCGTGGAG-3′, and REEP6_R1stop, 5′-AATCTAGAGCGGCCGCTCACTTGTCCTTCGGCTGCGGGGTCTGGC-3′. The two observed PCR amplicons corresponded to the predicted REEP6.1 and REEP6.2 sizes and were excised and gel purified (QIAquick Gel Extraction Kit, QIAGEN) prior to cloning into the pSC-B-amp/kan vector (StrataClone Blunt PCR Cloning Kit, Agilent Technologies). Clone identities (REEP6.1, GenBank: NM_001329556.1; REEP6.2, GenBank: NM_138393.2) were confirmed by Sanger sequencing (Source BioScience). Mutations were introduced by site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs) using primers and conditions specified by the NEBaseChanger software. Sequence integrity was confirmed by Sanger sequencing as before. To create expression vectors, wild-type (WT) and mutant REEP6.1 sequences were cloned into pEYFP-N1 (Clontech) digested with BmtI and NotI to release the EYFP sequence. WT and mutant REEP6.1 expression plasmids were transfected into SK-N-SH (ATCC) cells using TransIT-LT1 Transfection Reagent (Mirus Bio) using the manufacturers’ recommended conditions, in 8-well Nunc Lab-Tek Permanox chamber slides (ThermoFisher Scientific) plated at 40,000 cells/well and 6-well plates plated at 500,000 cells/well.

100 µL/well of freshly isolated PMN in RPMI-1640 medium at a concentration of 2 million/mL were seeded in Nunc™ Lab-Tek™ Permanox chamberslides (Thermo Fisher Scientific). The chamber slides were incubated with 50 pg/cell monosodium urate (MSU) crystals for 4 h at 37 °C to allow NET formation. The medium alone served as unstimulated control. Afterwards, samples were fixed using 2% paraformaldehyde for 20 min at RT. After washing of the samples three times with PBS, NETs/PMN were permeabilized using 0.1% Triton X-100 in PBS for 5 min at RT before blocking with 10% FCS, 2% BSA in PBS for 1 h at RT. Mouse anti-DNA-IgM (CBL186, Merck KGaA; diluted 1:100) and rabbit anti-human MPO antibody (1:100, ab9535, Abcam, Cambridge, UK, poly-clonal IgG) were incubated in blocking buffer ON at +4 °C. After washing three times with PBS, Alexa Fluor™ 568 goat anti-rabbit IgG (H+L) (1:400, ab175471, Abcam, Cambridge, UK, polyclonal IgG) and Cy™5 AffiniPure goat anti-mouse IgM (H+L) (1:400, 115-175-075, Jackson ImmunoResearch Europe Ltd.) in blocking buffer were used as a secondary antibody. The secondary antibodies were incubated with the DNA stains DAPI (ThermoFisher) and Sytox^TM Green (ThermoFisher) for 1 h at RT. After another washing step with two times PBS and one-time deionized water, the chambers were removed and slides were mounted using DAKO fluorescent mounting medium (Agilent).