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-1-alpha

AK8250-0005 5µg Ask for price

rHu MIP-1-alpha

AK8250-0020 20µg Ask for price

rHu MIP-1-alpha

AK8250-0100 100µg Ask for price

rHu MIP-1-alpha

AK8250-1000 1mg Ask for price

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 397
Description: Rabbit Polyclonal Antibody for MIP-3 alpha Antibody (CCL20) detection.tested for IHC in Human, Mouse.

MIP-3-alpha/CCL20, Human

HY-P7262 10ug
EUR 268

MIP-3-alpha/CCL20, Mouse

HY-P7263 10ug
EUR 234

MIP-3 alpha, CCL20, human

RC315-31 5ug
EUR 104.38
  • Product category: Proteins/Recombinant Proteins/Cytokines

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 294

MIP 1 alpha antibody

20R-1788 100 ug
EUR 673
Description: Rabbit polyclonal MIP 1 alpha antibody

MIP-1-alpha Antibody

5246-100
EUR 338

MIP-1 alpha Antibody

45363-100ul 100ul
EUR 252

MIP-1 alpha Antibody

45363-50ul 50ul
EUR 187

MIP-1 alpha Antibody

DF8572 200ul
EUR 304
Description: MIP-1 alpha Antibody detects endogenous levels of total MIP-1 alpha.

MIP-1 alpha Antibody

20-abx148975
  • EUR 425.00
  • EUR 342.00
  • 100 ug
  • 50 ug
  • Shipped within 5-10 working days.

MIP- 1 alpha Antibody

ABD8572 100 ug
EUR 438

Anti-MIP- alpha antibody

STJ94130 200 µl
EUR 197
Description: Rabbit polyclonal to MIP-1alpha.

Anti-MIP- alpha antibody

STJ98775 200 µl
EUR 197
Description: Rabbit polyclonal to MIP-3alpha.

MIP-3

EF016984 96 Tests
EUR 689

MIP-3

EF017888 96 Tests
EUR 689

MIP-3

EF013760 96 Tests
EUR 689

MIP-3

EF013761 96 Tests
EUR 689

MIP-3

EF016652 96 Tests
EUR 689

MIP-3

EF010865 96 Tests
EUR 689

MIP-3

EF012726 96 Tests
EUR 689

MIP-3

PAab09791 100 ug
EUR 386

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

Anti-MIP-3 alpha Antibody Biotin Conjugated

B00748-1 100ug
EUR 432
Description: Rabbit Polyclonal MIP-3 alpha Antibody Biotin Conjugated. Validated in WB and tested in Human.

MIP-3-alpha/CCL20 (CHO-expressed), Human

HY-P7260 10ug
EUR 268

MIP-3-alpha/CCL20 (HEK293-expressed), Rat

HY-P7261 10ug
EUR 268

Mouse MIP-3 alpha/CCL20 ELISA Kit

LF-EK50839 1×96T
EUR 648

Recombinant Human MIP-3 Alpha (CCL20) Protein

PROTP78556-2 20ug
EUR 317
Description: MIP-3α is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and can signal through the CCR6 receptor. MIP-3α 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-3α is an 8.0 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

Recombinant Murine MIP-3 Alpha (CCL20) Protein

PROTO89093-1 20ug
EUR 317
Description: MIP-3α is a CC chemokine that is expressed in the liver, lymph nodes, appendix, PBL and lung and signals through the CCR6 receptor. MIP-3α 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-3α is a 7.9 kDa protein containing 70 amino acid residues, including the four highly conserved cysteine residues present in CC chemokines.

Human MIP-3 alpha/CCL20 ELISA Kit

RK00207 96 Tests
EUR 521

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

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 VEGF-165

AK8295-0002 2µg Ask for price

rHu VEGF-165

AK8295-0010 10µg Ask for price

rHu VEGF-165

AK8295-0100 100µg Ask for price

rHu VEGF-165

AK8295-1000 1mg Ask for price

rHu EG-VEGF

AK8315-0005 5µg Ask for price

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 343

VEGF-121-Fc

E13-m005-2 50μg
EUR 1155

VEGF 121, CF

PR15029CF 5 ug
EUR 415

Human VEGF 121 Antibody

32562-05111 150 ug
EUR 261

Recombinant Human VEGF 121 Protein

RP00017 5 μg
EUR 174

Recombinant human VEGF 121 Protein

RP01162 5 μg
EUR 151

Human VEGF 121 Antibody (Biotin Conjugate)

32562-05121 150 ug
EUR 369

Human CellExp? VEGF 121, Human Recombinant

6484-10
EUR 343

Human CellExp? VEGF 121, Human Recombinant

6484-50
EUR 1333

Human VEGF 121 AssayLite Antibody (FITC Conjugate)

32562-05141 150 ug
EUR 428

Human VEGF 121 AssayLite Antibody (RPE Conjugate)

32562-05151 150 ug
EUR 428

Human VEGF 121 AssayLite Antibody (APC Conjugate)

32562-05161 150 ug
EUR 428

Human VEGF 121 AssayLite Antibody (PerCP Conjugate)

32562-05171 150 ug
EUR 471

Recombinant Human VEGF121 Protein

PROTP15692-10 10ug
EUR 317
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 317
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 545

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

QP5207-10ug 10ug
EUR 237

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

QP5207-1mg 1mg
EUR 3754

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

QP5207-2ug 2ug
EUR 136

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

QP5207-500ug 500ug
EUR 2322

VEGF Vascular Endothelial Growth Factor (121 a.a.) Mouse Recombinant Protein

PROTQ00731-2 Regular: 10ug
EUR 317
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.

VEGF Vascular Endothelial Growth Factor (121 a.a.) Human Recombinant Protein

PROTP15692-5 Regular: 10ug
EUR 317
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.

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

QP13933-10ug 10ug
EUR 201

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

QP13933-1mg 1mg
EUR 3573

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

QP13933-2ug 2ug
EUR 155

Superior Prime Broth™

121 500 ml
EUR 64

VEGF (121a.a.) Human, Vascular Endothelial Growth Factor (121) Human Recombinant Protein, HEK

PROTP15692 Regular: 10ug
EUR 317
Description: Recombinant Human VEGF 121 amino acids, produced in HEK cells is a glycosylated 37kDa homodimer and 50kDa homotrimer.;The VEGF is purified by proprietary chromatographic techniques.

VEGF Human, Vascular Endothelial Growth Factor (121 a.a.) Human Recombinant Protein, Sf9

PROTP15692-1 Regular: 10ug
EUR 317
Description: Vascular Endothelial Growth Factor-121 Human Recombinant produced in insect cells as an 18kDa homodimer, is a glycosylated, polypeptide chain containing 121 amino acids and having a molecular mass of approximately 36kDa.;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.

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