Nanodrop nd 1000 spectrophotometer

The isolation of the ribonucleic acid (RNA) from the CAFs and from the NFs was carried out with the RNeasy® Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The concentration and purity of the isolated RNA samples were determined with the NanoDrop® ND-1000 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). The reverse transcription was performed using the peqstar thermal cycler (Peqlab Biotechnologie GmbH, Erlangen, Germany). The complementary deoxyribonucleic acid (cDNA) was synthesized from the messenger RNA (mRNA) using the enzyme reverse transcriptase. The qPCR for analysis of gene expression was performed with PCR Real-Time CFX Connect (Bio-Rad Laboratories GmbH, Hercules, CA, USA) and normalized to β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference genes. The primers for Cav-1α, Cav-1β, β-actin, and GAPDH were designed using the Primer-BLAST program and chosen as follows: Cav-1α (NM_001753.4) forward, 5′-CAGAACAAACCTTTGGCGGG-3′ and reverse, 5′-CCTTCCTGGGCATGGAGTCCT-3′; Cav-1β (NM_001172896.1) forward, 5′-TCGGAGCGGTTAGTTCGATT-3′ and reverse, 5′-GGTTGACCAGGTCGATCTCC-3′; β-actin forward, 5′-GGAGCAATGATCTTGATCTT-3′ and reverse, 5′-CCTTCCTGGGCATGGAGTCCT-3′; GAPDH forward, 5′-AAAAACCTGCCAAATATGAT-3′ and reverse, 5′-CAGTGAGGGTCTCTCTCTTC-3′.

Protein concentrations of cell extracts were determined using the Bio-Rad Protein Assay Kit according to the manufacturer's instructions using BSA as the standard. The concentrations of purified proteins were obtained by the absorbance at 280 nm using the NanoDrop® ND-1000 Spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Alternatively, protein concentrations were determined using the Pierce® BCA Protein Assay Kit and BSA as the standard according to the manufacturer's protocol (Pierce® BCA^TM Protein Assay Kit, Thermo Scientific).

Agarose resin conjugated to anti-FLAG antibody M2 (A2220, Sigma) was used for protein purification. Proteins were extracted by main cultures inoculated and expressed as described above. Cells were harvested by centrifugation for 10 min at 4°C and 9,500 g, the cell pellet was re-suspended in lysis buffer (50 mM Tris/HCl pH 7.4, 100 mM NaCl and 1% TritonX) and lysed by sonication on ice. Cell lysates were clarified by centrifugation for 30 min at 4°C and 17,000 g and filtered with a syringe filter (0.45 μm). The proteins were purified using an agarose resin conjugated to anti-FLAG antibody M2 (A2220, Sigma) for 1.5 h, 4°C rotating. The resin was washed four times with 1 mL TBS for 5 min, 1,000 g, 4°C. Elution was carried out by 200 μl TBS containing 150 μg/ml of 3 × FLAG peptide (F4799, Sigma) and subsequently shaking at 4°C for 1 h. Further the resin was centrifuged at 8,200 g for 3 min and the supernatant was taken as protein solution. The concentrations of purified proteins were obtained by the absorbance at 280 nm using the NanoDrop^® ND-1000 Spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Total RNA was isolated from NK cells with the RNeasy Mini Kit (Qiagen, Hilden, Germany). For RNA quantification, a NanoDrop ND-1000 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany) was used. Complementary DNA synthesis was performed with the QuantiTect Reverse Transcription Kit (Qiagen). For quantitative reverse transcription polymerase chain reaction (qRT-PCR) either the Fast SYBR Green Master Mix (Applied Biosystems, Darmstadt, Germany) or the TaqMan Fast Universal PCR Master Mix (2X; Applied Biosystems) was used on a 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). Primers for NCR1, Fcer1g and CD247 were purchased from Qiagen or Thermofisher (Germany). For analysis, the expression levels of all target genes were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ΔCt). Gene expression values were calculated with the ΔΔCt method.

Plasmid DNA or PCR-products were purified using NucleoSpin Plasmid Kit or NucleoSpin Gel and PCR Clean-up Kit according to the supplier’s protocol (Macherey-Nagel). Quality and quantity of DNA was assessed with a peQLab Nanodrop ND-1000 spectrophotometer. For verification of cheA-mutants long range PCR was performed with Qiagen HotStarTaq DNA polymerase using Thermocycler 2720 Applied Biosystems, and primer pair cheA check B and sacB check A (Table 2). Custom DNA Sanger sequencing was performed by StarSEQ GmbH.

Colonized scaffolds (all four variants) were snap-frozen after 24 h, 7 days and 14 days (each n = 5) and homogenized in RLT-buffer (Qiagen, Hilden, Germany) with a tissue lyser (Qiagen) for 2 times each 3 min at 50 Hz. RNA was isolated and purified using the RNeasy Mini kit according to the manufacturer´s instructions (Qiagen), including on-column DNAse treatment. Purity and quantity of the RNA samples were monitored for RNA content and purity (260/280 absorbance ratio) using the Nanodrop ND-1000 spectrophotometer (Peqlab, Biotechnologie GmbH, Erlangen, Germany).

RNA was isolated in phase lock tubes using TRIzol (Invitrogen) according to the manufacturer’s protocol. To avoid genomic DNA contamination RNA was treated with Dnase I (Qiagen). Purified RNA was eluted in 20.0 μL of nuclease-free water and stored at −20.0 °C. RNA concentration and purity was assessed using a Nanodrop ND-1000 spectrophotometer (Peqlab). RNA (1.0 μg) was subjected to reverse transcription reaction using the high-capacity quantitect reverse transcription kit (Qiagen) according to the manufacturer’s protocol. Relative mRNA levels were quantified via real-time PCR (RT-qPCR) using a Bio-Rad C1000 qPCR Detection System and Power SYBR Green PCR Master Mix as recommended by the manufacturer (Life Technologies). All reactions were performed in triplicate from RNA isolated from three independent biological experiments.