BMDMs were reverse transfected with siRNAs against screen hit genes using Viromer Green (Lipocalyx, cat# VG-01LB-00). 5 μl of siRNAs (0.5 μM) were spotted in 96-well plates (BD Falcon, cat# 353219), and 0.1 μl of Viromer Green transfection reagent pre-mixed with 4.9 μl of Viromer Green Buffer was added to each well. Plates were tapped to mix the transfection reagents and centrifuged at 400 g for 1 s. After incubation for 30 min at room temperature, 40,000 BMDMs in 90 μl of complete DMEM were seeded per well for a final siRNA concentration of 25 nM. Plates were incubated at room temperature for 10 min to allow the cells to settle, then at 37°C in a humidified atmosphere with 5% CO2 for 48 hr. Cells were stimulated with 5 nM lipid A (Avanti Polar Lipids, Alabaster, AL, cat# 699500P) for time periods as indicated. Cells from 24 wells were pooled for total RNA extraction with RNeasy Mini Kit (Qiagen), then reverse transcribed with iScript cDNA synthesize kit (Biorad, cat# 1708891). For measurement of secreted protein level, supernatants were collected and subject to ELISA as described above. Selected siRNAs for the 4 hit genes were all from Thermofisher with the following siRNA IDs: s106096 and 298393 for Helz2; s104443 and s104445 for Phf11d, s100717 and 163480 for Sertad3, s76360 and s76361 for Zscan12.
Viromer green
Manufactured by BioNTech
Sourced in Germany
Viromer Green is a laboratory equipment product used for the efficient and gentle transfection of cells. It facilitates the introduction of genetic material, such as plasmids or small interfering RNAs, into a wide range of cell types. The product's core function is to enable effective and reliable delivery of these molecules into the target cells.
Automatically generated - may contain errors
Lab products found in correlation
On targetplus, by Horizon Discovery (2 mentions)
Myd88 sirna, by Thermo Fisher Scientific (2 mentions)
Tye fluorescent labeled transfection control sirna, by OriGene (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Viromer green, by BD (1 mentions)
Iscript cdna synthesize kit, by Bio-Rad (1 mentions)
Lipid a, by Avanti Polar Lipids (1 mentions)
Viromer green transfection reagent, by BioNTech (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Pierce gaussia luciferase glow assay kit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Celigo s imaging cytometer, by Revvity (1 mentions)
Scramble sirna, by Santa Cruz Biotechnology (1 mentions)
Sirna buffer, by Horizon Discovery (1 mentions)
Cellstar, by Greiner (1 mentions)
D 001810 10 20, by Horizon Discovery (1 mentions)
Smartpool, by Horizon Discovery (1 mentions)
On targetplus hspb1 sirna, by Horizon Discovery (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
15 protocols using viromer green
1
Functional Screening of Inflammatory Genes
2
Adipocyte Silencing of AS160/TBC1D4
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Fully differentiated ADMSCs-derived adipocytes were transfected using the Viromer GREEN transfection reagent (Lipocalyx, Halle, Germany) according to the manufacturer’s instruction. Briefly, siRNA targeting AS160/TBC1D4 at a concentration of 25 nM (Silencer Select Pre-designed siRNAs for AS160/TBC1D4—ID: s19140 and ID: s19142) and non-targeting control siRNA (Silencer Select Negative Control #1 siRNA) were used. The standard complexation protocol was employed using Viromer GREEN (Lipocalyx) according to the manufacturer's instruction.
3
siRNA Screening for MHV Replication Factors
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A custom siRNA library targeting each individual RTC-proximal factor (On Target Plus, SMART pool, 96-well plate format, Dharmacon, GE Healthcare) was ordered. Additionally, a deconvolved library of 4 individual siRNAs was purchased for selected targets. 10 nM siRNA were reverse transfected into L929 cells (8*103 cells per well) using Viromer Green (Lipocalyx) according to the manufacturer’s protocol. Cells were incubated 48 hr at 37°C 5% CO2 and cell viability was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). Cells were infected with MHV-Gluc (MOI = 0.05, 1000 plaque forming units/well), washed with PBS 3 h.p.i. and incubated in MEM+/+for additional 9 or 12 hr. Gaussia luciferase was measured from the supernatant using Pierce Gaussia Luciferase Glow Assay Kit (ThermoFisher Scientific). Experiments were carried out in four independent replicates and both cytotoxicity values and luciferase counts were normalized to the corresponding non-targeting scrambled control of each plate. A one-way ANOVA (Kruskal-Wallis test, uncorrected Dunn’s test) was used to test the statistical significance of reduced viral replication (mean <95% as compared to scramble control, n = 216). The R package ggplot2 was used to create the bubble plot (Figure 4b ).
4
siRNA-Mediated Silencing of HCAR3 in Monocytes
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Freshly isolated human monocytes were transfected with siRNA specifically targeting HCAR3 (OriGene), as shown in Stäubert et al. (2015) [50 (link)], using Viromer Green (Lipocalyx) according to the manufacturer’s protocol. 10 μL of siRNA-Viromer mix were preplated into a 96-well plate before the addition of freshly isolated monocytes resuspended in RPMI supplemented with 10% FBS (1.5 x 105 monocytes/well) which were then incubated 14 h at 37 °C in a humidified 5% CO2 incubator. cAMP assays were performed as described below. To monitor the transfection efficiency a red fluorescent siRNA (OriGene) and Hoechst 33342 (Sigma-Aldrich) were used. The viability of transfected monocytes was determined by staining with propidium iodide (PI) (Thermo Fisher Scientific) and Hoechst 33342. Transfection efficiency and viability were analyzed using the Celigo S Imaging Cytometer (Nexcelom). The transfection efficiency was found to be ≥ 80% with less than 50% of monocytes being PI-positive (S6 Fig ).
5
Knockdown Assay for VK2 Cell Response
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Transfection of VK2 cells with siRNA (ThermoFisher) was performed using Viromer Green (Lipocalyx) at 65% confluence. siRNAs used were as follows: s168 (TLR2), s14196 (TLR4), s2681 (CD44), s14199 (TLR5), AM4611 (negative siRNA 1). Transfection was performed at ×0.5 reaction volume (single knockdown) or ×0.25 reaction volume (double knockdown) following the manufacturer's protocol for 48 h, followed by challenge with HA and/or flagellin as previously described for 24 h.
6
SREBP1 Knockdown and T1317 Treatment
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Cells were seeded on either 12- or 96-well cell culture treated plates at a concentration of 1000,000 and 60,000 cells/well, respectively, and transfection experiment was performed as it was described earlier (8 (link)). The cells were transfected with final concentrations of 60 nM small interfering RNA for SREBP1 (Santa Cruz, # sc-36557) for 24 h using Viromer technology according to the manufacturer’s protocol (Viromer Green, Lipocalyx). Scramble siRNA (Santa Cruz, #sc-37007) was used as a control for the experiments. Knockdown of SREBP1 was confirmed by qPCR analysis 24 h post transfection. For further analysis, 24 h after transfection cells were either left untreated or treated with 2 μM T1317 as described earlier in our monocyte priming protocol. IL-6 and TNFα concentrations were analyzed as described in the respective paragraphs of the section “Materials and Methods.”
7
Macrophage siRNA Knockdown and Rescue
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iBMDMs were seeded at 5 × 104 cells/well in a tissue culture‐treated Nunc 24‐well plate and at 1 × 104 cells/well in a 96‐well plate (Greiner CELLSTAR®) in complete RPMI‐1640 containing 10% L929 cell supernatant and cultured overnight at 37°C. All siRNA stocks were reconstituted in siRNA buffer (Horizon Discovery) to 20 μM. SMARTpool ON‐TARGETplus siRNA was used for Dmxl1 (L‐055471‐01‐0005, Horizon Discover) alongside an ON‐TARGETplus non‐targeting pool as siRNA control (D‐001810‐10‐20, Horizon Discovery). Cells were transfected with siRNAs using Viromer Green (Lipocalyx) according to manufacturer instructions. Macrophages were transfected with siRNA at a final concentration of 50 nM. After 48 h culture, 96‐well plates were used for bead assays, while 24‐well plates were used for qRT–PCR analysis to confirm Dmxl1 mRNA knockdown. In Dmxl1 rescue experiments, Dmxl1 siRNA was co‐transfected with plasmids expressing either 3xFLAG‐DMXL1 (1773‐2047) or 3xFLAG‐DMXL1 S1903A/S1904A (1,773–2,047). Each well in a 24‐well and 96‐well plate was co‐transfected with 125 ng and 50 ng cDNA, respectively.
8
Transfection of Monocyte-Derived Dendritic Cells
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For microarray analysis, immunoblot and confocal imaging 3 × 106 MDDCs were transfected with 83 nM pre‐ or anti‐miR using the DC Nucleofection kit (Amaxa) according to the manufacturer's instructions (program U‐02). For flow cytometry, 106 MDDCs were transfected with 100 nM pre‐ or anti‐miR in a 12‐well plate using the Viromer GREEN (Lipocalyx). The recommended protocol for suspension cells was used.
9
Silencing Hsp27 in Monocytes
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Small interfering RNA was delivered into total monocytes using Viromer technology according to the manufacturer’s protocol (Viromer Green, Lipocalyx). Monocytes were transfected with either control or Hsp27/HSPB1 siRNA (SMARTpool, ON-TARGETplus HSPB1 siRNA, Dharmacon) for 24 h. Knockdown of Hsp27 was confirmed by Immuno- blotting. For downstream analysis, transfected cells at 200,000 cells/ml were either left untreated, stimulated with 100 ng/ml LPS alone for 3 h or followed by 20 mins incubation with 300 μM BzATP. IL-1β production was measured by real-time qPCR, immuno-blotting and ELISA.
10
MyD88 and Smad7 Knockdown Protocols
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