Retinal samples (n = 4–8/group) were analyzed for the mRNA expression of specific markers (Table 2 ). mRNA was isolated and reverse transcribed using the MultiMACS mRNA and cDNA Synthesis Kit on the MultiMACS™ M96 Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. RT-qPCR was performed with 40 cycles using the Universal SYBR Green Supermix (Biorad, Hercules, CA, USA). A total of 5 µL of cDNA (concentration 1 ng/µL) was used in a volume of 20 µL reaction mix. cDNA expression levels of investigated genes were normalized to the housekeeping genes β-ACTIN (ATCB, SI) and RLP4. The final primer concentration was 100 nM.
Multimacs m96 separator
Manufactured by Miltenyi Biotec
Sourced in Germany
The MultiMACS™ M96 Separator is a magnetic separation device designed for high-throughput cell separation and purification. It enables efficient and automated processing of up to 96 samples simultaneously, using magnetic beads or particles. The device provides a standardized and reproducible separation process, ensuring reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Universal sybr green supermix, by Bio-Rad (2 mentions)
Multimacs mrna and cdna synthesis kit, by Miltenyi Biotec (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Lysis buffer, by Miltenyi Biotec (1 mentions)
Thermocycler, by Bio-Rad (1 mentions)
Streptavidin microbeads, by Miltenyi Biotec (1 mentions)
4 protocols using multimacs m96 separator
1
Retinal mRNA Expression Analysis
2
Inflammatory and Barrier Marker Expression
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The expression of inflammation markers, like NF-κB, TNF-α, IL-1β and NLRP3, was analyzed. The mRNA expression of tight junction’s marker occludin and the glycocalyx marker galectin-3 was also investigated in the porcine model (Table 2 ). Briefly, after incubation, the corneas were cut into small pieces and incubated in 800 µL Lysis Buffer (Miltenyi Biotec, Köln, Germany) for 1 h at 37 °C on a shaker. The mRNA was isolated from porcine cornea explants and reverse transcribed using the MultiMACS mRNA and cDNA synthesis was done with cDNA Synthesis Kit on the MultiMACS™ M96 Separator (Miltenyi Biotec, Köln, Germany) according to the manufacturer’s protocol. After cDNA synthesis, a quantitative real-time PCR (qRT-PCR) was performed using the SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, Feldkirchen, Germany) in a thermal cycler (Bio-Rad CFX96™ Real-Time System, Bio-Rad, Feldkirchen, Germany), as described previously [56 (link)]. β-actin (ACTB) and Ribosomal protein L 4 (RPL4) were used as housekeeping genes.
3
Retinal Explant Gene Expression Analysis
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The expression of cell specific markers, like parvalbumin (PVALB), CD11b and CC‐chemokine ligand 2 [CCL2] were analysed. Markers of oxidative stress, including iNOS, hypoxia‐inducible factor (HIF)‐1α and heat shock protein 70 (HSP70), were also investigated in the retinal organ model. mRNA was isolated from retinal explants and reverse transcribed using the MultiMACS mRNA and cDNA Synthesis Kit on the MultiMACS™ M96 Separator (Miltenyi Biotec) according to the manufacturer's protocol. After cDNA synthesis, qRT‐PCR was performed with 40 cycles using the Universal SYBR Green Supermix on a thermocycler (Biorad). About, 1 ng/µL of cDNA was used in a reaction volume of 20 µL according to the manufacturer's instructions. Final primer concentration was 100 nmol/L. The cDNA expression levels of the investigated genes were normalized to the cDNA level of the housekeeping genes RLP4 and β‐actin. The primers (Table 2 ) were designed using the Primer3 software (GenBank: KM035791.1, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ ).
4
Yeast-Displayed Antibody Selection
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Yeast library selections were performed as previously described7 ,10 . Briefly, the Exo201 (acute COVID-19 cohort) or Exo204 (BRI, Yale HCW, CoronaVac, longitudinal control, and myocarditis cohorts) yeast library was induced at an OD of 1 cultured in 1:10 SDO-Ura:SGO-Ura at 30 C. Prior to selection, 58 induced yeast were set aside to allow for comparison of the pre-selection to post-selection libraries. 108 induced yeast were washed with PBE and added to wells of a sterile 96-well plate. Ten micrograms of yeast adsorbed IgG was added to the yeast library in duplicate in 100 uL PBE and incubated for 1 h at 4 C. Yeast were washed with PBE and incubated with 1:100 biotin anti-human IgG Fc antibody (clone HP6017, BioLegend #409308, or clone QA19A42, Biolegend #366918) for 30 min. Yeast was washed with PBE and incubated with a 1:20 dilution of Streptavidin MicroBeads (#130-048-101, Miltenyi Biotec) for 30 min. Yeast was resuspended in PBE and IgG-bound yeast was isolated by positive magnetic selection using the MultiMACS M96 Separator (Miltenyi Biotec) according to manufacturer instructions and as previously described7 ,10 . Selected yeast was resuspended in 1 mL SDO -Ura and incubated at 30 °C for 24 h.
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