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NANO-100 micro-spectrophotometer

Manufactured by Allsheng
Sourced in China

The NANO-100 micro-spectrophotometer is a compact and precise instrument designed for the measurement of absorbance and transmittance spectra of small sample volumes. It features a wavelength range of 190 to 1100 nanometers and a wavelength accuracy of ±0.5 nanometers. The NANO-100 is equipped with a high-performance detector and a user-friendly software interface for data acquisition and analysis.

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6 protocols using NANO-100 micro-spectrophotometer

Total RNA from bEnd.3 cells were isolated with an RNA isolator (Vazyme, Nanjing, China). The concentration of RNA was measured by a NANO-100 micro-spectrophotometer (ALLSHENG, China), and the absorbance at 260/280 nm was considered to detect the purity of RNA. HiScript® Q RT SuperMix for qPCR (Vazyme, Nanjing, China) was used to synthesize cDNA from RNA. Subsequently, qRT-PCR was performed with ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) on LightCycler 480 II (Roche, Basel, Switzerland). mRNAs in the tested samples were standardized with Actb, and the 2ΔΔCt method was used for relative quantification. Primer sequences are listed in Table S1.
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The mRNA of markers of M2 macrophages and glycolysis hub gens were measured by qRT-PCR. Total RNA was separated using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The quality and concentration of RNA were determined using a Nano-100 micro-spectrophotometer (Allsheng, China). cDNA was reverse-transcribed from RNA using PrimeScript RT Master Mix (TaKaRa, Japan). For expression analysis, PCR was performed with TB Green™ Premix Ex Taq™ II (TaKaRa, Japan) in a Mx3000P Real-Time fluorescence quantitative PCR system (Agilent, USA), and 18SrRNA was used as an internal control. Changes were determined using the 2−ΔΔct calculation method, and all measurements were performed in triplicate. See Table 1 for primer information.

Primers information

Gene namePrimers sequencesSpecies
18SrRNAF: 5ʹ-ATGCGGCGGCGTTATTCC-3’Mouse
F: 5ʹ-GCTATCAATCTGTCAATCCTGTCC-3’ 
IFI27F: 5ʹ-GACTCTCCGTGCCATCTACTG-3’Mouse
 R: 5ʹ-CCTCTATCGCCATATCTGCCAC-3’ 
CD163F: 5ʹ- CTGGCGGGTGGTGAAAACA-3’Mouse
R: 5ʹ- CAGCCGTTACTGCACACTG-3’ 
CD206F: 5ʹ- GAGGGAAGCGAGAGATTATGGA-3’Mouse
R: 5ʹ- GCCTGATGCCAGGTTAAAGCA-3’ 
HK2F: 5ʹ- TGATCGCCTGCTTATTCACGG-3’Mouse
R: 5ʹ- AACCGCCTAGAAATCTCCAGA-3’ 
LDHAF: 5ʹ-TGTCTCCAGCAAAGACTACTGT-3’Mouse
R: 5ʹ-GACTGTACTTGACAATGTTGGGA-3’ 
PGK1F: 5ʹ-ATGTCGCTTTCCAACAAGCTG-3’Mouse
R: 5ʹ-GCTCCATTGTCCAAGCAGAAT-3’ 
ENO1F: 5ʹ-TGCGTCCACTGGCATCTAC-3’Mouse
 R: 5ʹ-CAGAGCAGGCGCAATAGTTTTA-3’ 
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RNA isolator (Vazyme, Nanjing, China) was used following the manufacturer’s protocol to extract the total RNA from bEnd.3 cells. The purity and concentration of isolated RNA were determined with a NANO-100 microspectrophotometer (ALLSHENG, Hangzhou, China), and an equal amount of RNA was reverse-transcribed into cDNA using an HiScript® Q RT SuperMix for qPCR (Vazyme, Nanjing, China). Real-time PCR was then performed on LightCycler 480 II (Roche, Switzerland) with the primers and ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). The mRNA levels were normalized against Actb and quantified using the 2−ΔΔCt method. Primer sequences are listed in Table S1.
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To quantitatively analyze gene expression, total RNA was extracted from cells or tumor tissue using TRIZOL reagent (Ambion) following the manufacturer’s instructions. The RNA was treated with DNase and the concentration was measured by a NANO-100 Micro Spectrophotometer (ALLSHENG). Complementary DNA was synthesized using a PrimeScript RT regent kit with gDNA Eraser (TaKaRa) with 1 μg of total RNA following the manufacturer’s protocols. Quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix Ex Taq II (TaKaRa) with a QuantStudio 6 Pro Real-Time PCR system (Thermo Fisher Scientific). The data were expressed as relative mRNA levels and normalized to the 18S rRNA as previously described (35 (link)). The sequence of the primers is shown in Table S2.
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Resting and Activated Mature CD8 + T cells
Resting and activated mature CD8þ T cells were harvested and washed once in phosphate-buffered saline (PBS). DNA preparation was performed using a High Pure PCR Template Preparation Kit (Indianapolis, Indiana). Briefly, 200 μl of sample material was added to 200 μl of binding buffer and 40 μl proteinase K. The resulting mixture was incubated at 70°C for 10 min. After adding 100 μl isopropanol, the sample was loaded into high filter tubes. After centrifugation, 500 μl inhibitor removal buffer was added. The samples were centrifuged and washed twice with 500 μl wash buffer. Prewarmed elution buffer was added in order to elute the DNA. The concentration of DNA was calculated using a Nano-100 Micro-spectrophotometer (Allsheng, Hangzhou city, China).
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Total RNA from bEnd.3 cells was isolated with RNA isolater (Vazyme, Nanjing, China). The concentration of RNA was measured by NANO-100 micro-spectrophotometer (ALLSHENG, China) and the absorbance at 260/280 nm was concerned to detect the purity of RNA. HiScript® Q RT SuperMix for qPCR (Vazyme, Nanjing, China) was used to synthesize cDNA from RNA. Subsequently, qRT-PCR was performed with ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) on LightCycler 480 II (Roche, Basel, Switzerland). mRNA in the tested samples were standardized with Actb, and the 2 -ΔΔCt method was used for relative quanti cation. Primer sequences were listed in Supplement Table 1.
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