Monitoring SARS-CoV-2 Growth in Cell Culture

A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts.
Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity.
Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses.
IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics.
Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms.
In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.
pharmacodane

pharmacodane

rHu MIP-3-beta
AK8232-0005 5µg Ask for price

rHu MIP-3-beta
AK8232-0020 20µg Ask for price

rHu MIP-3-beta
AK8232-0100 100µg Ask for price

rHu MIP-3-beta
AK8232-1000 1mg Ask for price

rHu MIP-4
AK8218-0002 2µg Ask for price

rHu MIP-4
AK8218-0010 10µg Ask for price

rHu MIP-4
AK8218-0100 100µg Ask for price

rHu MIP-4
AK8218-1000 1mg Ask for price

rHu MIP-1-beta
AK8264-0002 2µg Ask for price

rHu MIP-1-beta
AK8264-0010 10µg Ask for price

rHu MIP-1-beta
AK8264-0100 100µg Ask for price

rHu MIP-1-beta
AK8264-1000 1mg Ask for price

rHu TNF-alpha
AK8324-0010 10µg Ask for price

rHu TNF-alpha
AK8324-0050 50µg Ask for price

rHu TNF-alpha
AK8324-0100 100µg Ask for price

rHu TNF-alpha
AK8324-1000 1mg Ask for price

rHu GRO-alpha
AK9903-0005 5µg Ask for price

rHu GRO-alpha
AK9903-0025 25µg Ask for price

rHu GRO-alpha
AK9903-0100 100µg Ask for price

rHu GRO-alpha
AK9903-1000 1mg Ask for price

Anti-MIP-3 alpha Antibody
A00748 100ul
EUR 476.4
Description: Rabbit Polyclonal Antibody for MIP-3 alpha Antibody (CCL20) detection.tested for IHC in Human, Mouse.

MIP-3 alpha Recombinant Protein
40-353-0005mg 0.005 mg
EUR 311.1
Description: MIP-3 alpha is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and signals through the CCR6 receptor. MIP-3 alpha is chemotactic towards lymphocytes and dendritic cells. Additionally, it promotes the adhesion of memory CD4+ T cells and inhibits colony formation of bone marrow myeloid immature progenitors. Recombinant murine MIP-3 alpha is a 7.9 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

MIP-3 alpha Recombinant Protein
40-353-002mg 0.02 mg
EUR 437.1
Description: MIP-3 alpha is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and signals through the CCR6 receptor. MIP-3 alpha is chemotactic towards lymphocytes and dendritic cells. Additionally, it promotes the adhesion of memory CD4+ T cells and inhibits colony formation of bone marrow myeloid immature progenitors. Recombinant murine MIP-3 alpha is a 7.9 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

MIP-3 alpha Recombinant Protein
40-380-0005mg 0.005 mg
EUR 311.1
Description: MIP-3 alpha is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and can signal through the CCR6 receptor. MIP-3 alpha is chemotactic towards lymphocytes and dendritic cells. Additionally, it promotes the adhesion of memory CD4+ T cells and inhibits colony formation of bone marrow myeloid immature progenitors. Recombinant human MIP-3 alpha is an 8.0 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

MIP-3 alpha Recombinant Protein
40-380-002mg 0.02 mg
EUR 437.1
Description: MIP-3 alpha is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and can signal through the CCR6 receptor. MIP-3 alpha is chemotactic towards lymphocytes and dendritic cells. Additionally, it promotes the adhesion of memory CD4+ T cells and inhibits colony formation of bone marrow myeloid immature progenitors. Recombinant human MIP-3 alpha is an 8.0 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

MIP-3-alpha/CCL20, Human
HY-P7262 10ug
EUR 321.6

MIP-3-alpha/CCL20, Mouse
HY-P7263 10ug
EUR 280.8

MIP-3 alpha, CCL20, human
RC315-31 5ug
EUR 125.26

Ccl20 Antibody / Mip-3 alpha
R32858 100 ug
EUR 419

rHu SDF-1-alpha
AK8318-0002 2µg Ask for price

rHu SDF-1-alpha
AK8318-0010 10µg Ask for price

rHu SDF-1-alpha
AK8318-0100 100µg Ask for price

rHu SDF-1-alpha
AK8318-1000 1mg Ask for price

rHu TNF-alpha (cGMP)
AK9897-0000 1mg Ask for price

rHu IFN-alpha-2A
AK9909-0020 20µg Ask for price

rHu IFN-alpha-2A
AK9909-0100 100µg Ask for price

rHu IFN-alpha-2A
AK9909-1000 1mg Ask for price

rHu IFN-alpha-2B
AK9910-0020 20µg Ask for price

rHu IFN-alpha-2B
AK9910-0100 100µg Ask for price

rHu IFN-alpha-2B
AK9910-1000 1mg Ask for price

Anti-MIP-3 Alpha/CCL20 Antibody
A00748-2 100ug/vial
EUR 352.8

MIP-1 alpha Antibody
45363-100ul 100ul
EUR 302.4

MIP-1 alpha Antibody
45363-50ul 50ul
EUR 224.4

MIP 1 alpha antibody
20R-1788 100 ug
EUR 807.6
Description: Rabbit polyclonal MIP 1 alpha antibody

MIP-1 alpha Antibody
20-abx148975
  • EUR 510.00
  • EUR 410.40
  • 100 ug
  • 50 ug

MIP- 1 alpha Antibody
ABD8572 100 ug
EUR 525.6

MIP-1 alpha Antibody
DF8572 200ul
EUR 420

Anti-MIP- alpha antibody
STJ94130 200 µl
EUR 236.4
Description: Rabbit polyclonal to MIP-1alpha.

Anti-MIP- alpha antibody
STJ98775 200 µl
EUR 236.4
Description: Rabbit polyclonal to MIP-3alpha.

MIP-1-alpha Antibody
5246-100 each
EUR 405.6

rHu IFN-alpha-2B (cGMP)
AK8237-0000 1mg Ask for price

rHu IFN-alpha-2A (cGMP)
AK8561-0000 1mg Ask for price

rHu SDF-1-alpha (cGMP)
AK9881-0000 1mg Ask for price

MIP-3
PAab09791 100 ug
EUR 463.2

MIP-3
EF013760 96 Tests
EUR 826.8

MIP-3
EF013761 96 Tests
EUR 826.8

MIP-3
EF016652 96 Tests
EUR 826.8

MIP-3
EF016984 96 Tests
EUR 826.8

MIP-3
EF017888 96 Tests
EUR 826.8

MIP-3
EF010865 96 Tests
EUR 826.8

MIP-3
EF012726 96 Tests
EUR 826.8

rHu MCP-3
AK8261-0002 2µg Ask for price

rHu MCP-3
AK8261-0010 10µg Ask for price

rHu MCP-3
AK8261-0100 100µg Ask for price

rHu MCP-3
AK8261-1000 1mg Ask for price

rHu IL-3
AK8321-0002 2µg Ask for price

rHu IL-3
AK8321-0010 10µg Ask for price

rHu IL-3
AK8321-0100 100µg Ask for price

rHu IL-3
AK8321-1000 1mg Ask for price

rHu NT-3
AK8543-0002 2µg Ask for price

rHu NT-3
AK8543-0010 10µg Ask for price

rHu NT-3
AK8543-0100 100µg Ask for price

rHu NT-3
AK8543-1000 1mg Ask for price

rHu Galectin-3
AK9079-0010 10µg Ask for price

rHu Galectin-3
AK9079-0050 50µg Ask for price

rHu Galectin-3
AK9079-0100 100µg Ask for price

rHu Galectin-3
AK9079-1000 1mg Ask for price

rHu Eotaxin 3
AK9970-0005 5µg Ask for price

rHu Eotaxin 3
AK9970-0020 20µg Ask for price

rHu Eotaxin 3
AK9970-0100 100µg Ask for price

rHu Eotaxin 3
AK9970-1000 1mg Ask for price

Anti-MIP-3 alpha Antibody Biotin Conjugated
B00748-1 100ug
EUR 518.4
Description: Rabbit Polyclonal MIP-3 alpha Antibody Biotin Conjugated. Validated in WB and tested in Human.

Mouse MIP-3 alpha/CCL20 ELISA Kit
LF-EK50839 1×96T
EUR 777.6

MIP-3-alpha/CCL20 (CHO-expressed), Human
HY-P7260 10ug
EUR 321.6

MIP-3-alpha/CCL20 (HEK293-expressed), Rat
HY-P7261 10ug
EUR 321.6

Expression of ADR-α1, 2 and ADR-β2 in cumulus cellculture of infertile women with polycystic ovary syndrome and poor responder who are a candidate for IVF: the novel strategic role of clonidine in this expression

Objective: Alpha and beta-adrenoceptors (ADR-α1, 2, and β2) play a regulatory role in the folliculogenesis and steroidogenesis in the ovarian follicles. This study aimed to measure these adrenoceptors mRNA and its protein levels in cumulus cells (CCs) culture of poor ovarian reserve (POR) and polycystic ovarian syndrome (PCOS) infertile women (IVF candidate) and the effect of clonidine treatment at CCs culture.
Methods: This case/control study was conducted in 2017 includes a control (donation oocytes) and two studies (PCO and POR) groups. The ovulation induction drugs were prescribed in all groups. After the oocyte puncture, the follicular fluid was collected and CCs were isolated were cultured. RNA was extracted and cDNA was synthesized and designed the primer for the ADR-α1, 2 and ADR-β2 gene expression. The protein levels were investigated by Western Blot.
Results: The results showed a high level of three adrenergic expressions in PCO women compared to the control group (p-value <.001), which can be reduced by clonidine. POR group showed a significant decrease in the gene expression of ADR-α1 (p-value = .004) and ADR-α2 (p-value = .003) compared to the control group and clonidine treatment had no effect.
Conclusion: The significant increase of three adrenoceptors gene expression and protein levels in CCs culture indicate to the hyperactivity of the ovarian sympathetic nervous system at the receptor levels in women with PCOS, and clonidine confirmed it by reducing this expression. In POR women, the reduction of ADR-α1, 2 expressions maybe lead to the aging process in the ovary.

rHu EG-VEGF
AK8315-0020 20µg Ask for price

rHu EG-VEGF
AK8315-0100 100µg Ask for price

rHu EG-VEGF
AK8315-1000 1mg Ask for price

rHu VEGF (cGMP)
AK9063-0000 1mg Ask for price

VEGF-121-Fc
E13-m005-1 10μg
EUR 411.6

VEGF-121-Fc
E13-m005-2 50μg
EUR 1386

VEGF 121, CF
PR15029CF 5 ug
EUR 498

Human VEGF 121 Antibody
32562-05111 150 ug
EUR 313.2

VEGF 121 Recombinant Protein
96-768 0.02 mg
EUR 519
Description: Vascular endothelial growth factor (VEGF) is also known as vascular permeability factor (VPF) and VEGF-A, and is a member of the platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family and encodes a protein that is often found as a disulfide linked homodimer. This protein is a glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis. Alternatively spliced transcript variants, encoding either freely secreted or cell-associated isoforms, have been characterized. Alternatively spliced isoforms of 121,145,165,183,189 and 206 amino acids in length are expressed in humans. VEGF165 appears to be the most abundant and potent isoform, followed by VEGF121 and VEGF189. VEGF121 is the only form that lacks a basic heparinbinding region and is freely diffusible. Mouse embryos expressing only the corresponding isoform (VEGF120) do not survive to term, and show defects in skeletogenesis. Human VEGF121 shares 87% aa sequence identity with corresponding regions of mouse and rat, 93% with feline, equine and bovine, and 91%, 95% and 96% with ovine, canine and porcine VEGF, respectively. VEGF121 induces the proliferation of lymphatic endothelial cells. The lymphangiogenesis may be promoted by upregulation of VEGF121, which may in turn act in part via induction of VEGF-C.

Recombinant human VEGF 121 Protein
RP01162 5 μg
EUR 181.2

Recombinant Human VEGF 121 Protein
RP00017 5 μg
EUR 208.8

Human VEGF 121 Antibody (Biotin Conjugate)
32562-05121 150 ug
EUR 442.8

Human CellExp? VEGF 121, Human Recombinant
6484-10 each
EUR 411.6

Human CellExp? VEGF 121, Human Recombinant
6484-50 each
EUR 1599.6

Human VEGF 121 AssayLite Antibody (FITC Conjugate)
32562-05141 150 ug
EUR 513.6

Human VEGF 121 AssayLite Antibody (RPE Conjugate)
32562-05151 150 ug
EUR 513.6

Human VEGF 121 AssayLite Antibody (APC Conjugate)
32562-05161 150 ug
EUR 513.6

Human VEGF 121 AssayLite Antibody (PerCP Conjugate)
32562-05171 150 ug
EUR 565.2

Recombinant Human VEGF121 Protein
PROTP15692-10 10ug
EUR 380.4
Description: VEGF is a potent growth and angiogenic cytokine. It stimulates proliferation and survival of endothelial cells, and promotes angiogenesis and vascular permeability. Expressed in vascularized tissues, VEGF plays a prominent role in normal and pathological angiogenesis. VEGF signals through three receptors; fms-like tyrosine kinase (flt-1), KDR gene product (the murine homolog of KDR is the flk-1 gene product) and the flt4 gene product. Due to its increased acidity, VEGF121 circulates more freely than other VEGF forms, which bind more tightly with vascular heparin sulfates. Recombinant human VEGF121 is a 28.4 kDa disulfide-linked homodimeric protein consisting of two 121 amino acid polypeptide chains.

VEGF (121 a.a.), Vascular Endothelial Growth Factor-121 Human Recombinant Protein, His Tag
PROTP15692-8 Regular: 10ug
EUR 380.4
Description: Vascular Endothelial Growth Factor-121 Human Recombinant produced in E.Coli is a double, non-glycosylated, polypeptide chain (aa 207-327) containing a total of 142 amino acids and having a molecular mass of 16.3 kDa. ;The VEGF-121 is fused to a 20 amino acid His tag at N-terminus and purified by proprietary chromatographic techniques.

Recombinant Human VEGF-121 Isoform Protein, Untagged, E.coli-100ug
QP5207-100ug 100ug
EUR 654

Recombinant Human VEGF-121 Isoform Protein, Untagged, E.coli-10ug
QP5207-10ug 10ug
EUR 284.4

Recombinant Human VEGF-121 Isoform Protein, Untagged, E.coli-1mg
QP5207-1mg 1mg
EUR 4504.8

Recombinant Human VEGF-121 Isoform Protein, Untagged, E.coli-2ug
QP5207-2ug 2ug
EUR 163.2

Recombinant Human VEGF-121 Isoform Protein, Untagged, E.coli-500ug
QP5207-500ug 500ug
EUR 2786.4

VEGF Vascular Endothelial Growth Factor (121 a.a.) Human Recombinant Protein
PROTP15692-5 Regular: 10ug
EUR 380.4
Description: Vascular Endothelial Growth Factor-121 Human Recombinant produced in E.Coli is a double, non-glycosylated, polypeptide chain containing 121 amino acids and having a molecular mass of 28423 Dalton. VEGF121 circulates more freely than other VEGF forms, which bind more tightly with vascular heparin sulfates.;The VEGF-121 is purified by proprietary chromatographic techniques.

VEGF Vascular Endothelial Growth Factor (121 a.a.) Mouse Recombinant Protein
PROTQ00731-2 Regular: 10ug
EUR 380.4
Description: Vascular Endothelial Growth Factor-121 Mouse Recombinant produced in E.Coli is a homodimer, non-glycosylated, polypeptide chain containing 121 amino acids and having a molecular mass of 28.4 kDa.;Recombinant Mouse VEGF-121 is a truncated version of Murine VEGF-165.;The VEGF-121 is purified by proprietary chromatographic techniques.

Recombinant Mouse VEGF (121 a. a. ) Protein, Untagged, E.coli-10ug
QP13933-10ug 10ug
EUR 241.2

Recombinant Mouse VEGF (121 a. a. ) Protein, Untagged, E.coli-1mg
QP13933-1mg 1mg
EUR 4287.6

Recombinant Mouse VEGF (121 a. a. ) Protein, Untagged, E.coli-2ug
QP13933-2ug 2ug
EUR 186

Sheep polyclonal antibody for Herpes Simplex virus
121 1 ml
EUR 327.53
Description: This is sheep polyclonal antibody against Herpes Simplex virus type 2 for WB, ELISA.

rHu Insulin
AK8211-0025 25mg Ask for price

rHu Insulin
AK8211-0250 250mg Ask for price

rHu Insulin
AK8211-1000 1g Ask for price

rHu aFGF
AK8230-0010 10µg Ask for price

rHu aFGF
AK8230-0050 50µg Ask for price

rHu aFGF
AK8230-0100 100µg Ask for price

rHu aFGF
AK8230-1000 1mg Ask for price

rHu MCSF
AK8238-0002 2µg Ask for price

rHu MCSF
AK8238-0010 10µg Ask for price

rHu MCSF
AK8238-0100 100µg Ask for price

rHu MCSF
AK8238-1000 1mg Ask for price

rHu CTGF
AK8240-0005 5µg Ask for price

rHu CTGF
AK8240-0020 20µg Ask for price

rHu CTGF
AK8240-0100 100µg Ask for price

rHu CTGF
AK8240-1000 1mg Ask for price

rHu MIF
AK8255-0005 5µg Ask for price

rHu MIF
AK8255-0025 25µg Ask for price

rHu MIF
AK8255-0100 100µg Ask for price

rHu MIF
AK8255-1000 1mg Ask for price

rHu Myostatin
AK8256-0002 2µg Ask for price

rHu Myostatin
AK8256-0010 10µg Ask for price

rHu Myostatin
AK8256-0100 100µg Ask for price

rHu Myostatin
AK8256-1000 1mg Ask for price

rHu Leptin
AK8265-0200 200µg Ask for price

rHu Leptin
AK8265-1000 1mg Ask for price

rHu Leptin
AK8265-5000 5mg Ask for price

rHu RANTES
AK8275-0005 5µg Ask for price

rHu RANTES
AK8275-0020 20µg Ask for price

rHu RANTES
AK8275-0100 100µg Ask for price

rHu RANTES
AK8275-1000 1mg Ask for price

rHu BDNF
AK8286-0002 2µg Ask for price

rHu BDNF
AK8286-0010 10µg Ask for price

rHu BDNF
AK8286-0100 100µg Ask for price

rHu BDNF
AK8286-1000 1mg Ask for price

rHu CNTF
AK8302-0005 5µg Ask for price

rHu CNTF
AK8302-0020 20µg Ask for price

rHu CNTF
AK8302-0100 100µg Ask for price

rHu CNTF
AK8302-1000 1mg Ask for price

rHu EGF
AK8327-0100 100µg Ask for price

rHu EGF
AK8327-0500 500µg Ask for price

rHu EGF
AK8327-1000 1mg Ask for price

rHu GDNF
AK8335-0002 2µg Ask for price

rHu GDNF
AK8335-0010 10µg Ask for price

rHu GDNF
AK8335-0100 100µg Ask for price

rHu GDNF
AK8335-1000 1mg Ask for price

rHu OSM
AK8531-0002 2µg Ask for price

rHu OSM
AK8531-0010 10µg Ask for price

rHu OSM
AK8531-0100 100µg Ask for price

rHu OSM
AK8531-1000 1mg Ask for price

rHu BAFF
AK8540-0005 5µg Ask for price

rHu BAFF
AK8540-0020 20µg Ask for price

rHu BAFF
AK8540-0100 100µg Ask for price

rHu BAFF
AK8540-1000 1mg Ask for price

rHu Artemin
AK9076-0005 5µg Ask for price

rHu Artemin
AK9076-0020 20µg Ask for price

rHu Artemin
AK9076-0100 100µg Ask for price