Nanodrop lite

HEK001 cells were seeded in 6-well plates at 250,000 cells/well in 2 mL of medium (keratinocyte serum-free medium and l-glutamine (99:1)). The cells were incubated at 37 °C in 5% CO₂ and a 95% air-humidified atmosphere for 24 hr. After 24 h of incubation, the cells were treated with 6.59 µM of D. The compound was tested in duplicate and cells with medium without D were used as a control (n = 3). The exposure period was 24 h.
After the incubation time, the total RNA from each sample was extracted and purified from HEK001 cells using a RNeasy^® Plus Mini insolation Kit (Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. Total RNA concentration and quality were spectrophotometrically measured using the absorbance ratio 260:280 nm with NanoDrop^TM Lite (ThermoFisher Scientific, Waltham, MA, USA). Once the RNA was obtained, cDNA was synthesized with PxE Thermal Cycler (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s recommendations. For microarray analysis, GeneChip^® Clariom S Human Array (ThermoFisher Scientific, Waltham, MA, USA) was used. Data were generated and processed with Affymetrix software (Santa Clara, CA, USA). Gene expression data from the samples were compared using a one-way t-test using stringent transcript cut-off criteria with fold change (FC) > 1.5 and p-value ≤ 0.05.

Genomic DNA from two million total CD4⁺ T cells was isolated using the Gentra Puregene cell kit (Qiagen GmbH, Hilden, Germany) and the DNA was measured using a NanoDrop^TM Lite (Thermofisher Scientific, Waltham, MA, USA). An Alu-based quantification of HIV-1 proviral load was performed on genomic DNA using primers encompassing a conserved region in the HIV-1 genome (LTR region) and genomic Alu sequences ProF (5′-AGT AGA TGC TAC GTA ACG TGC TGA ACC CAC TGC TTA AGC CTC) and Alu22R (5′-CTG GGA TTA CAG GCG TGA G) at a concentration of 25 nM each [48 (link),49 (link),50 (link),51 (link)]. One microliter aliquots of the outer PCR were subjected to an inner qPCR reaction using primers PVLF (5′-AGT AGA TGC TAC GTA ACG TG) and PVLR (5′-AAG GGT CTG AGG GAT CTC) at a concentration of 25 nM each. Each qPCR reaction was performed in a 10 µL total reaction volume containing Kapa SYBR^® FAST mix buffer. The qPCR conditions were 94 °C for 5 min, 40 cycles of 94 °C for 5 s, 57 °C for 15 s, and 63 °C for 45 s. All qPCRs were performed in a 384-well plate format using the LightCycler 480 II (Roche, Basel, Switzerland). In addition, serial dilutions of genomic DNA of an 8E5 cell line (with one HIV-1 copy per cell) generated a standard curve that enabled the quantification of the number of HIV-1 copies per cell in the DNA samples.

Bacterial DNA was isolated from fecal samples using the QIAamp^® Fast DNA Stool Mini Kit (QIAGEN^®, Germany) automated on a QIAcube^® (QIAGEN), according to manufacturer’s protocol with the addition of a manual pretreatment step to enhance lysis of gram positive bacteria. In this pretreatment step, fecal samples containing storage buffer were centrifuged for 5 min at 14,500 x g, the supernatant was discarded, and all samples, including frozen samples, were resuspended in InhibitEX^® lysis buffer. Bead beating was performed using a single 5 mm stainless steel ball (QIAGEN) on a TissueLyser LT (QIAGEN) for 4 min at 30 Hz. This was followed by lysis at 95 °C for 5 min, and 200 μL of the resulting lysate solution was transferred to spin columns continuing with the standard protocol.
Purity of the extracted DNA was evaluated spectrophotometrically, with a Nanodrop^TM Lite (Thermo Fisher Scientific) using the A_260/280 OD ratio. The concentration of DNA was measured using the Qubit^TM HS Assay (Thermo Fisher Scientific). Finally, DNA integrity was evaluated by agarose gel electrophoresis.

Approximately 70–80 mg of hypothalamus and hippocampus tissues were dissected from each rat brain, transferred to cell disruption buffer and homogenized with 1.5 mm homogenization beads (Triple-Pure™, Molecular Biology Grade Zirconium Beads) using the BeadBug microtube homogenizer. After homogenization, RNA extraction was performed according to the manufacturer’s protocol using the PARIS™ kit (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The RNA was quantified using a Nanodrop™ spectrophotometer (Nanodrop Lite; Thermo Fisher Scientific). RNA (500 ng) was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit, and qPCR was performed using the Bio-Rad real-time system CFX96 to amplify gene expression levels with the following parameters: 40 cycles of 2 min at 50 °C, 5 min at 95 °C, 10 s at 95 °C and 30 s at 60 °C. mRNA levels were normalized to D-Box, and fold changes were calculated using the 2^−ΔΔCt method. The primers used to amplify these genes are listed in Supplementary Table S1.

WBC was isolated from blood by hemolysis of red blood cells with 0.83% ammonium chloride followed by washing twice with phosphate buffer saline (PBS). Total amount of DNA was extracted from WBCs by using QIAamp DNA Mini Kit (51304, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After measurement of DNA concentration of WBC DNA by a spectrophotometer NanoDropLite (Thermo Fisher Scientific, Waltham, MA, USA). Primers to amplify the envelope or pX region of BLV were used for nested PCR according to the protocols by Fechner et al.^{36 (link)} and Murakami et al.^{37 (link)}. PCR was carried out in a total reaction volume of 20 µl containing 0.5 U of polymerase from GoTaq Hot Start Green Master Mix (M5122, Promega, Madison, WI, USA) or SapphireAmp Fast PCR Master Mix (RR350A, Takara Bio, Kusatsu, Japan), 0.5 µM of forward and reverse primers, and 1 µl of extracted WBC DNA (100–400 ng). Thermal cycling condition was as follows: 95 °C for 2 min, followed by 35 cycles of 94 °C for 45 s, 62 °C for 30 s, 72 °C for 30 s, and finally 72 °C for 4 min.

The methods employed for RNA extraction and amplification were adapted from Martha et al. [8 (link)]. Blood was collected from the jugular vein at three different time points: immediately prior to MCAO surgery, 5 minutes after MCA reperfusion, and 72 hours post-MCAO procedure. Sham rats underwent the complete 5t-MCAO procedure, with the exception that the filament was not placed to induce a stroke; blood samples were taken at the same time points for sham animals. Total RNA was extracted from the pellet/buffy coat using the Nucleospin Blood Kit (Macherey-Nagel, Düren, Germany). RNA quantity was assessed with a Nanodrop Lite (Thermo-Fisher; Waltham, MA). cDNA synthesis utilized the RT² First Strand Kit from Qiagen, and gene expression analysis of 84 genes was performed using the ABI StepOne Plus Qiagen (Germantown, MD) and the RT² Profiler Rat Chemokine and Receptor Array from Qiagen.