Zirconia silica bead

Transcription levels of pro-inflammatory cytokine genes, IL-1β (5′-CACCTCTCAAGCAGAGCACAG-3′ and 5′-GGGTTCCATGGTGAAGTCAAC-3′), IL-6 (5′-TCCTACCCCAACTTCCAATGCTC-3′ and 5′-TTGGATGGTCTTGGTCCTTAGCC-3′), and immunosuppressive cytokine IL-10 (5′-AGTCAGCCAGACCCACAT-3′ and 5′-GGCAACCCAAGTAACCCT-3′) were determined as previously described [47 (link)]. In brief, the frozen colon and brain tissues in RNA preservative solution were homogenized by using 1 mm sterile zirconia/silica bead (Biospec Products, Bartlesville, US) and Minibeadbeater (Biospec Products, Bartlesville, US). Next, homogenized tissues were extracted from RNA using TRI reagent (TRIzol® Reagent, Ambion, Life Technologies, CA, US) according to the recommendations of the manufacturer. Then, a DNase treatment was performed by adding the DNA removal and inactivation kit (Ambion, Life Technologies, CA, US). The extracted tissue RNA was converted to complementary DNA (cDNA) using reverse transcription reagents (Tetro cDNA synthesis kit, Bioline, US). SYBR-Green (SensiFAST SYBR Lo-ROX kit, Bioline, US)-based real-time quantitative PCR was conducted using the primers and further analyzed by comparative Ct method. The mRNA expression levels of target genes were normalized with Gapdh (5′-GTATTGGGCGCCTGGTCACC-3′ and 5′-CGCTCCTGGAAGATGGTGATGG-3′) mRNA levels.

The antibiotic
susceptibility of biofilm cells was determined by following the same
procedure described in our previous studies.^{23 (link),30 (link)} Briefly, rSMPs with attached biofilm cells were washed three times
with 0.85 wt % NaCl solution and transferred to a 40 °C prewarmed
test tube containing 2 mL of 0.85 wt % NaCl solution. After incubation
for 10 min, the sample was transferred to a test tube at room temperature
containing 0.85 wt % NaCl solution. Three cycles of temperature change
were applied. For the programmed rSMPs, biofilm cells detached by
shape recovery were harvested upon the completion of the third cycle.
Biofilm cells on flat rSMPs were harvested by bead-beating for 30
s by using 0.1 g of 0.1 mm zirconia/silica bead (BioSpec Products,
Inc., Bartlesville, OK). To avoid the confounding effect of bead-beating,
the same bead-beating was also applied to the biofilm cells detached
by shape recovery. The harvested biofilm cells from both the programmed
rSMP and the static control were then treated with 50 μg/mL
tobramycin (Tokyo Chemical Industry Co., Tokyo, Japan) for 1 h at
37 °C and washed three times with 0.85 wt % NaCl solution. The
washed samples were plated on LB agar plates to count colony forming
units (CFU)^{31 (link)} and antibiotic susceptibility
was determined by comparing them to the controls.

5–40 ml of yeast culture was centrifuged and resuspended in 1 mL cold TCA 20%. Cells were concentrated to 200 μL in 10% TCA, and 200 μL glass beads (Zirconia/Silica Beads; BioSpec Products, Inc., Bartlesville, OK) was added. Cells were then lysed using a Bullet Blender Storm 24 (Next Advance, Inc., Averill Park, NY). Supernatants were transferred to new tubes, beads washed twice with 200 μL TCA 10%, and extracts were pooled. Extracts were centrifuged at 3000 rpm for 10 min, and the pellet was resuspended in 30–200 μL Laemmli buffer containing 5–10 μl Tris base (1 M). The extracts were boiled 5 min, centrifuged 5 min at 12,000 rpm and transferred to a new tube. Protein concentration was determined by Bradford assay.

DNA extraction was performed as previously described with modifications (Turnbaugh et al., 2009 (link)). Briefly, 300 μl aliquot of cultures with starch as sole carbon source was mixed with a solution containing 500 μl of extraction buffer (200 mM Tris pH 8.0, 200 mM NaCl, 20 mM EDTA), 200 μl of 20% SDS, 500 μl of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) and 1.2 mg of 0.1-mm diameter zirconia/silica beads (BioSpec Products, United States). The suspension was then subjected to 3 min of bead beating (BioSpec Products, United States) at room temperature (RT), spun at 8,000 rpm for 5 min at RT, and then 750 μl of the top layer was transferred to a 15 ml tube (BD Falcon 12 × 75 mm, #352063) for immediate column purification (NucleoSpin, Macherey-Nagel, Switzerland). Column binding buffer NTl was used at 2.5 vol., 3 washes with washing buffer NT3 were performed and final elution was done with 25 μl of low T₍₁₀₎E_(0.1) buffer.