157 was added to the suspension and the mixture was subsequently centrifuged at 16,000 xg for 5 min. The bacterial pellet was suspended in 100 μl streptavidin-coated magnetic beads which was 10-fold diluted in PBS/EDTA and incubated on ice for 20 min, followed by another washing step with 1 ml PBS/EDTA and centrifugation at 16,000 xg for 5 min. Subsequently, the bacterial pellet was suspended in 500 μl PBS/EDTA. MS Columns were placed in the MACS Separator (magnetic plate) and were pre-rinsed with 500 μl PBS/EDTA. Next, 500 μl cell suspensions were applied onto a MS Column. Unlabeled cells were collected by washing the MS Columns three times with 500 μl PBS/EDTA. After this step, MS Columns were removed from the separator and placed on the new collection tube. In order to collect the magnetically labeled cells, 1.06 ml PBS/EDTA was applied onto the MS Column and immediately flushed out. The obtained liquids were stored at -20°C for DNA extraction and flow cytometry (FC) analysis. Flow cytometry (FC) A flow cytometry analysis was performed to confirm the efficiency of the MACS procedure. To do so, 50 μl of the magnetic bead-labeled fraction was resuspended in 100 μl Anti-Human IgG (γ-chain specific)-FITC antibody (Sigma-Aldrich, St. Louis, MO, USA) which was 100-fold diluted in PBS and incubated on ice in the dark for 30 min. This suspension was washed with 1 ml PBS and centrifuged at 16,000 xg for 5 min. The bacterial pellet was resuspended in 50 μl PBS while adding 10 μl propidium iodide (PI) (0.1 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA). Fifty (50) μl of autologous serum-incubated stool supernatant suspension was labeled with Anti-Human IgG-FITC and another 50 μl was only incubated with PI as experimental controls. Samples were analyzed using the BDAccuriTM C6 flowcytometer (BDBiosciences, San Jose, CA, USA). Supernatant of three fecal samples with the largest bacterial pellet were washed with PBS and measured by flow cytometry to check the PI-induced background signal. The FL3-A signal above the threshold of these samples was measured in the analysis. Polymerase chain reaction (PCR) amplification and 16S rRNA gene sequencing Fecal microbial composition was determined by Illumina MiSeq paired-end sequencing of the V3-V4 hypervariable region of the 16S rRNA gene (MiSeq Bentchtop Sequencer, Illumina Inc., San Diego, USA). Amplification of bacterial DNA was performed by PCR using modified 341F and 806R primers with a six-nucleotide barcode on the 806R primer for multiplexing.18,19 Both primers contain an Illumina MiSeq adapter sequence, which is necessary for flow cell binding in the MiSeq machine. Primer sequences can be found in Supplementary Table S1. A detailed description of the PCR amplification procedure, DNA clean-up and MiSeq library preparation using a 2x300 cartridge can be found in the Supplementary Methods. Demultiplexing and clustering of sequencing reads was performed using Quantitative Insights In Microbial Ecology (QIIME) with UCLUST v.1.2.22q at 97% similarity.20,21 Taxonomic profiling was performed using Paired-eND Assembler for DNA sequences (PANDAseq).22 All sequences with quality scores <0.9 were discarded by PANDAseq to increase sequence read-out quality, and read numbers per sample were rarefied to 25,000 read/sample. QIIME was used to assign bacterial taxonomy down to family and genus level, and ARB was used to identify sequences further down to species level.23 Antimicrobial IgG immune responses in IBD
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