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Viral Infection - Science topic
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Questions related to Viral Infection
Working on viral infectious disease to evaluate different microRNAs profile using RT-PCR. I used mostly SV40 miRNA that's commonly used as an internal control, but sometimes in literature other internal controls used as U6, RNU44, U1, B-actin ,GAPDH etc.
Which type of internal control has the best accuracy for normalization instead of SV40?
Hello, I am currently establishing a virus infection model in my lab. The cell I use is a vero cell and the virus is porcine epidemic diarrhea virus (PDEV). I plan to use the virus stock without concentration titration to make the new virus stocks, and I will conduct TCID50 analysis of the virus stocks in the future.
First, 0.5 ug/ml to 2 ug/ml TPCK-trypsin test was conducted in 96 well for the condition of virus infection culture (including 0.3% BSA). The virus infection culture was treated after washing twice with plain DMEM using 80-90% vero cells (Figure 1). Then, the appropriate concentrations (0.5 ug/ml, 0.75 ug/ml, 1 ug/ml) were selected and PEDV infection was performed.
Similar to the above process, after washing twice with plain DMEM, the virus stock of unknown concentration was diluted by virus infection culture (by TPCK-trpysin concentration) at 1:10. Finally 5 ml was dispensed into 100 dishes for 1 hour incubation. Next, I washed it twice with plain DMEM, added 10ml of the virus-infected culture medium, and incubated it for 3 days (Figure 2).
What I am curious about is the concentration (0.5 ug/ml or 0.75 ug/ml?) of TPCK-trypsin to establish a virus infection model and the timepoint of harvesting cell supernatant for virus stock manufacturing. What state should the vero cell be in to manufacture the virus stock? Please advise us to establish a virus infection model. Thank you!


Hello,
I would like to ask which is the best terminology to use for describing a negative control in an experiment (drug testing in-vitro, viral infection): NC (negative control) or NTC?
Thank you for being so helpful.
Also, I recently saw several articles use the term naive to describe the negative control for non-infected animals.
Hello every one
Can any one suggest me trends subjects in viral infection in cancer patients for PHD thesis?
Thanks in advance.
I am having difficulty trying to increase the concentration of both low molecular and high molecular weight Poly I:C without increasing the volume. Can anyone help?
Hi,
I have Illumina RNA sequencing data for viral infection only or viral treated. I would quantify the viral gene expression, but the total reads counts is various between library. Please see fig. I am wondering do I need to downsample the total reads to 5 M or is it ok to quantify using the total viral mapped reads ?? Thanks in advance

Hello
How can i get my article :
Mly Ismail El Karimi, Khalid Hattaf, Noura Yousfi, Dynamics of an immunological viral infection model with lytic and non-lytic immune response in presence of cell-to-cell transmission and cure of infected cells, Commun. Math. Biol. Neurosci., 2022 (2022), Article ID 119
in ResearchGate
Thank you
I have two DEG sets for 2 disease conditions (from mild to severe condition) of the same viral infection. When I look at the common gene from these two sets of DEGs, I found that some genes show opposite expression among these two conditions ( Like a gene downregulated in mild but up-regulated in severe or vice versa). So what I want to know is that,
1) If this phenomenon is normal in viral infection??
Which viral infection might be more severe?a transported virus from an animal to a human or a virus directly invade human?
Hi everyone, i'm having problems with the MTT assay i'm performing, since two weeks.
I'm performing viral infection on Beas2B cells and a dose response curve of compounds with citotoxicity in parallel.
When i add MTT on the infected cells the wells get completely purple, while on the non-infected (citotoxicity) I see normal metabolism inside the cells.
What could be the reason? Do any of you have experimented something like this?
Hi all
I've been struggling with this problem for a few months now. I am transfecting a BMI1 overexpression plasmid with GFP (https://www.addgene.org/21577/) into Phoenix cells for retroviral production. The Phoenix cells are GFP+ 24 hours post transfection so I know that step is working.
However, when I try to collect the media (which presumably contains virus) and infect target cells using polybrene, GFP is not expressed up to 4 days post transduction. I cannot figure out what's going on. A test infection of 293T cells also did not express GFP so I suspect the problem is with viral production?
What is the best way to isolate extracellular vesicles from cell culture media if they are infected with viruses?
I am talking about an experiment in which we want to study the components inside the exosome after viral infection. I want to make sure that the isolation is clean of viruses.
If you mention the kit and the solutions, I will be grateful to you.
Crimean Congo hemorrhagic fever is an acute febrile viral disease with high mortality rate, the detection of this disease is through PCR for detection viral nucleic acid and ELISA to detect antibodies specific to the virus, carriers among human are uncommon and there is no many researches about human carriers.
My PI has given me a protocol for viral infection with HAZARA virus using Opti-mem media. The protocol calls for replacing the growth media with Opti-mem and adding the virus stock directly, incubating this for 90 minutes at 37°C with intermittent swirling, then removing this media and replacing with 2% supplemented DMEM. We have been mainly using HEK and Vero cells and getting no results. Could Opti-mem be interfering with the viral infection?
ADAM17 activity is rapid and reversible. Want to check if ADAM17 activated after viral infection in my cell line (Huh 7.5, A549) and if I can detect that by flow cytometry.
Can anyone share any detail protocol how I can detect activated ADAM17 on cell surface by flow cytometry?
We are having trouble measuring viral genome copy numbers using qPCR. The CT values for dengue virus are quite varied; between technical replicates (same sample), we may see 10 CT differences and sometimes no detection. Has anyone had a similar experience and found a way to correct the issue?
We speculate it may be generating cDNA using Random Primers. We usually use oligo dT for cDNA synthesis but switched with viral infection to random primers. Previously, we have had no issues with qPCR and have gotten consistent CT values.
The Random primers we use are a stock concentration of 10uM, added in 1ul volume to a 20 ul cDNA reaction. This is the concentration recommended by the kit (https://www.abmgood.com/onescript-plus-cdna-synthesis-kit.html).
Any tips would be greatly appreciated!
Hello
I am working on viral infection in PK-15 cells and then IFA in 96 well plates. After fixing the cells with 50%methanol and acetone for overnight at 40C and IFA, the wells turn completely white. Can anyone suggest what can be done for IFA in 96 well plates? I have also attached pictures herewith

Egypt has the highest prevalence of HCV. What is the major risk factor or history behind it?
IFN detection is hard in vivo and I would like to use a reporter to quantify IFN during a viral infection in vivo using flow. The Locksley lab has been reported to have a week signal so I would love to get suggestions on what other reporter mice to use.
I'm looking for published or unpublished papers.
We are looking for some viral disease on Tuberose, what do you think of this symptom?
The main goal is detection of Tuberose mild mosaic virus, please check this paper to compare symptoms:

I made an cytokines expression curve, in 7dpi and 14 dpi Interferon gamma have a expression peaks, but at 28 dpi I observed an expression below the mock / control. Could this result be only related to the resolution of the viral infection, or a possible way of evading the immune response?
I'm working with primary human cell lines and I'm having difficulty with a viral infection using lentivirus with a plasmid backbone ~13kb. I use a positive control, lentiGFP-NEO, which infects well but my experimental plasmid does not.
Other technical things I've tried:
- I use 4ug/ml of polybrene at the time of the infection.
- I've tried just applying the viral media directly to the cells and also have tried a spinfection
- I've also tried adding PEG-IT when harvesting the virus.
The positive control that I use for infection is the same positive control that I use for transfection. I've been carrying it through the extent of the experiment, which makes me think that my viral production (I make virus in HEKT 293Ts) is not the source of the problem.
Any and all suggestions would be appreciated!
We found many cases of covid-19 infection who suffer second time within 2-3 months after the first infection. It indicates the status of immunity of the infection which in most cases of other viral infection that imparts long time immunity even life long immunity. It is same type of immunity like influenza infection. Effectivity of influenza vaccine usually last for 6 months due to changing its antigenicity. So this vaccine lost its popularity in most of the countries particularly in developing countries and manufacturers failed to make business from this vaccine. In the same way, I am afraid that Covid-19 vaccine will fail to make much money of the manufacturer in few months and no more than 6 months.
I am performing an experiment analyzing the cytokine production of macrophages in response to viral infection.
I recently harvested and cultured peritoneal macrophages from mice. I plated these cells on 12-well plates and have been changing the media enriched with 1:1000 mCSF every two days. Once the cells reached 80-90% confluent I washed the cells two times with PBS and infected these cells with an inoculum of virus at an MOI of 3. After 1hr incubation, I washed the cells again two times with PBS and put 750uL of media back on cells. After 24hrs, I collected this 750uL of media to be used for 36-spot Luminex.
I'm concerned that I placed too much media on the cells after infection - do you think I have diluted the supernatant too much and my cytokines will all be below the limit of detection when I run a Luminex assay?
Thank you for your help.
Olivia
Hi,
I'm trying to stain a viral infection of the pancreas in paraffin sections of adult (3 month old) BALB/c mice.
Prior to starting my histology analysis, I have titrated the organ to confirm that I actually have a viral load in the pancreas of my infected animals.
I have also used a organ (liver) were I know that I have abundancy of viral positive cells as a control for my work.
In my protocol I did Citrate Buffer retrieval (I have also tried Tris-EDTA) on 97C, used a commercial block of endogenous HRP, and 3%BSA to block nonspecific binding. My primary antibody is a nuclear marker, and is biotinylated (I have also tried un-biotinylated). My secondary antibody S-POD (also tried Envision for un-biotinylated Ab) and I use DAB as my chromogen.
When developing my chromogen I observed little no-specific staining, mostly in the areas that have dried. However, I did observed a lot of small brown DAB spots that mostly fade once I counterstain. And the biggest problem is that I didn't observe a single positive nuclear staining in 3 separated experiments. In each experiment I used pancreases from 3 different mice.
I observed a sufficient viral titer, which meant that the virus is present in the pancreas and specific positive cells with no background staining in my positive control which meant that the protocol and my work was ok?!
I know that the maybe I should think about changing my viral marker, however, this marker is mostly abundantly present in other tested organs.
I was wondering if the pancreas as a organ has some specific guidelines that I must follow, or if someone has some tips or tricks for pancreas staining in IHC!
Thank you for your help and suggestions,
Fran
We're suddenly unable to get a good protein content after cell lysis preparation. I mean, almost no protein by coomasie blue staining of the gel, yet our protein standards/ladders show up fine. Previously, I had no problems with overall protein content or detecting desired bands with my protocols. We've purchased new RIPA buffer and protease inhibitors; varied lysis time, sonication, boiling, loading buffer...everything we can think of and none of it has made a difference. Does anyone have any ideas? Something I'm missing? Coinciding with the onset of this problem are other cellular changes that suggest our mice may be harboring a retrovirus. Is it possible that a viral protease is untouched by our inhibitor cocktail and be chewing up the proteins in our samples? I did try lysing with boiling laemmli buffer to overcome that possibility; it didn't help, but I may not have performed that correctly.
Any ideas or thoughts?
Hello,
I am trying to measure the cytokine storm markers following SARS-Cov-2 viral infection of lung cells and macrophages under different conditions. However, finding a safe way to deactivate the virus while preserving the markers is not straightforward to find in literature, any suggestions?
Although infection with coronavirus regarded as viral infection, however, its effect on immunity may lead to decreased immunity of the human body and lead to opportunistic infection, so maybe an introduction of potent antibiotics like 4th generation cephalosporin to be followed by other common usual antibiotics which currently used could have a potent effect in increase the improvement rate, especially for those with abdominal symptoms.
Hello, I am looking for a computational simulator of in vitro infection of cells by virus, to be used for teaching purposes. Do you know any tools/resources ? I would like students to set infection conditions, such as MOI, time, temperature and could visualize outcomes of infections as cytophatic effect, for example.
We usually do actual infections in the lab, but we are looking for a tool to perform this activities also in the virtual platform of the course.
Thank you very much in advanced
Kind regards.
If NAT(nucleic acid test) shows negative and CT shows positive, which should we believe?
Can we calculate their degrees of confirmation to make decision?
There are many confirmation measures. Are they practical?
Are they helpful for diagnosing viral infection?
The attached figure shows that NAT has lower sensitivity (0.5) and higher specificity (0.95); CT has higher sensitivity (0.80) and lower specificity (0.75).
My effort: https://www.mdpi.com/1099-4300/22/4/384

The adaptive, or acquired, immune response takes days or even weeks to become established—much longer than the innate response; however, adaptive immunity is more specific to pathogens and has memory. Adaptive immunity is an immunity that occurs after exposure to an antigen either from a pathogen or a vaccination. This part of the immune system is activated when the innate immune response is insufficient to control an infection. In fact, without information from the innate immune system, the adaptive response could not be mobilized. There are two types of adaptive responses: the cell-mediated immune response, which is carried out by T cells, and the humoral immune response, which is controlled by activated B cells and antibodies. Activated T cells and B cells that are specific to molecular structures on the pathogen proliferate and attack the invading pathogen. Their attack can kill pathogens directly or secrete antibodies that enhance the phagocytosis of pathogens and disrupt the infection. Adaptive immunity also involves a memory to provide the host with long-term protection from reinfection with the same type of pathogen; on re-exposure, this memory will facilitate an efficient and quick response.
How much favourable is -6.78 delta G value for docking a ligand for an enzyme, please help me with this as it will help me intern to create in-silico evidence for a viral infection.
I am not fully aware of all investigators.
Lymphocytes are a type of white blood cell that play several roles in the immune system, including protection against bacteria, viruses, fungi, and parasites. Lymphocyte increased in response to infections or cancer. However, in covid-19 infection the lymphocyte decreased.
Immunoglobulin is used to treat viral infection. There are different immunoglobulin already used for the treatment of different viral diseases. Whether immunoglobulin is available or not against the corona virus.
I have two groups of viral sequences classified by year of isolation. How do I compare between these groups to determine any genetic differences that may have contributed to an increase in the infection rate of the virus?
I have used the statics to define the diseased from Non diseased. however this comes by improving the process of laboratory diagnosing viral infection. These to best of knowledge , independently were done which cause redundancy and noise in the final rests , are they ever possible factors to include to have the true decision rather than cut-off point inclusion and exclusion since it is precedent by type I and type II errors any suggestions!
I am using IgM Mac ELISA for diagnosis of viral infection in our laboratory. I am strictly following the protocol as per the instruction from the manufacturer, but unfairly getting the high absorbance value in negative control. Have tried with all trouble shooting and even changed the kit batch number too. So can anyone help me out in rectifying the issue and solve it.
So, I can't wrap my head around the logic behind these calculations. I've figured out 2 methods, but they give me different answers. So I have 40 wells (80, 000 cells each), and I need 300 uL of virus (MOI 1) in each well.
Stock Concentration=1.81 x 10^9 PFU/mL; 5 uL of this is kept in each eppendorf
Method 1:
1.81 x 10^9 PFU/mL = 1.81 x 10^6 PFU/uL = 9.02 x 10^6 PFU/ 5 uL
So, we have 9.02 x 10^6 virions in each eppendorf.
And so, if we take the 5 uL of stock and add it to 2257.5 uL, we essentially end up with 9.02 x 10^6 virions in 2262.5 uL. This means that the concentration has become 4000 PFU/ uL. And so, 20 uL of this diluted virus contains 80 000 virions.
So, then I added 20 uL of this diluted virus and topped it off with 280 uL of media. This step is what confuses me. I understand that this may dilute the virus more, but don't I still have 80 000 virions in each well regardless? So the MOI is still 1, and shouldn't the cells still be infected?
Method 2:
I need 80 000 virions per well, with 40 wells. So, in total I need 3.2 x 10^6 virions. I have 1.81x10^6 PFU/uL stock. So, 3.2/1.81= 1.768 uL. So, this means i need 1.768 uL of stock. I have 40 wells which need 300 uL media each = 12 000uL media total.
Hence, I need 12 000-1.768= 11,998.232 uL of media added with 1.768 uL of stock concentration virus, to give me 12 000 uL of diluted virus with 80 000 virions for each 300 uL.
I already performed my infection with method 1, but I feel that it is wrong. Method 2 seems to make sense. Can anyone confirm and explain the correct logic behind it? Thanks.
What are the data needed and exclusion criteria for the researches studying the prevalence of infectious diseases or viral infection incidence in epidemiological researches?
How to design the study and what are the suggestions to get a good questionnaire form?
Regards;
I am trying to generate a stable cell line using lentiCRISPRv2 infection with my desired gRNA. This plasmid has the Puromycin selection marker. I used 2ug/mL Puromycin for selecting cells, but some cells in control group (No viral infection) are still present even after 3days of Puromycin containing media. I changed the media every 24 hrs. Can anyone guide in this how these cells are becoming resistant?
Hi, I am doing my macrophages culture from Porcine PBMCs of 3 months old piglets, So is it necessary that checking of maternally derived antibodies of a particular virus in that species? Again we have to give viral infection to macrophages after its complete growth and then harvest the cells. So please suggest me checking of MDA in piglets serum by cELISA is necessary?
I have read many papers when the author refer to a MOI value, I know what MOI means but I dont know what is a good MOI
the potential risk for Reye’s syndrome among children and teenagers when Infection with viral diseases and take Aspirin is taken as a hypothermia
I have 4 X 10 E10 PFU of my viral stock and I have to follow a protocol for the viral infection which suggests a MOI of 0.01, diluting the virus in 40 mL of BHI for infection per O157:H7 (containing 1.5 X 10 E8 cells). I tried to make calculations but I'm not sure they are correct. How many microliters do I have to use from my viral stock?
Hi,
Currently i am performing QC on new RNA-seq data. My fastq files were generated by Paired-end sequencing with a read length of 151 bases. Total rna was extracted and depleted of rRNA. RNA was isolated from a viral infection experiment. Upon looking at the fastqc output of the un-trimmed fastq files, most of the parameters were satisfied except for those mentioned above. I did get a warning for over represented sequences but these were found to be sequences related to the virus genome due to cellular replication.
My question is, that is this issue due to adapters of more likely a subsequence of the experiement conditions. I have attached pictures of output for failed modules.
Thanks.
Hello,
I would like to detect viral infection in may plant tissue cultures. I know there is Elisa tests and RT-PCR methods but all of them are refered to one specific virus. Is there any methods to screen my plants and after positive outcome make decision about virus identification or to throw out same cultures. I would be grateful for any sugestions.
It seems that viral infections have been described for virtually every human organ except for Bone. Certainly there are bony sequelae of viral infections, such as osteomyelitis variolans seen in smallpox, but I am not aware of a viral osteomyelitis per se. Is it possible that some pathologic or radiographic findings, may be explained by an as yet undescribed viral osteomyelitis?
My question refers to how to prevent viral infection by the pulverization of potential mice fecal material / urine on the leaf litter that is been examined.
Our lab has a frozen stock solution of polyinosinic:polycytidylic acid (poly IC). It's been in our -20C freezer since 2004. We've used it on and off since then. The concentration is 2.5mg/mL
I recently defrosted it for a cell culture. But once it became a solution, I saw a transparent mass in the Eppendorf. It looks like a small piece of plastic wrap/Ziploc bag. I tried to disperse it using a P1000 pipette, but the clump would not dissolve
I looked at the literature, and no paper mentions a precipitate.
Does anyone know if this clump is normal? Or should I get a new source of poly IC?
Thanks very much.
what amount of viral titre is being used for infecting c.elegans?
Can we use the processing waste of shrimp affected with viral infection, in particular WSSV, in aquafeed formulation?
Following the Zika virus epidemic in the Americas, a large proportion of the population has likely been immunized to Zika virus. Kawiecki et al reported DENV-2 enhancement by Zika virus-induced antibody response in vitro. Are there arguments that can make us expect more severe dengue outbreak in the Americas in the coming years?
Explanation:
After storing viral stocks for too long at -80oc are used to infect (MOI) the suitable cell line show's less infection as compared to the freshly prepared viral stock. What does it indicate? some of the virus particles in the stock get either dead, disappear or weak, as we are aware virus are called as biological puzzles outside the host they behave as dead and can stay too long even for years and while in inside their host they get activated and start replicating? Why is this decrease in infectivity when stored for too long?
Thanks.
I am looking for virus inactivation to discern the effect caused by virus replication itself and the effect caused by the already induced IFN by the virus infection (prsent in the vehicle of the virus).
I irradiated with 240nm UV to vanish any replicative form from my supernatant. Now I am wondering if the irradiation also inactivated the IFN present in the supernatant. I checked, with the available lamp in our lab, that cells just die when cultured with UV-irradiated media.
I want to use influenza A virus rescued from plasmids to infect MDCK cells. Could polybrene can improve the efficiency of the infection of influenza A virus?
Could anyone recommend protocol for production of infectious Varicella zoster virus (VZV) on MRC-5 cells?
I am using RetroNectin to coat plates for viral infection. The protocol (see the link below) says to block with 2% BSA for 30 min and wash with PBS after removing the RetroNectin coating. Does anyone know why these steps are needed, and would it affect the viral infection efficiency if not block/wash?
Also, the protocol says to spin down the virus first, and then add cells. Would it make a big difference if I spin the virus and the cells together? I am afraid that I would lose some virus if spin down the virus first and remove the supernatant.
Thanks for your help!
RetroNectin protocol link:
Varicella zoster virus is the cause of chickenpox and zoster often produce a highly contagious and mild disease in children but if an adult acquires a primary VZV infection, it may lead to more severe disease.
I am trying to understand the expression of Fc receptor on microglia post viral infection. Hence, I am not adding Fc Block in the cells. However, as a control should I use Fc Block in the isotype? I assume that I should add Fc Block in the isotype controls otherwise my sample and isotype may show the same signal? Any suggestion?
Many scientists talked about first report of VNN in marine fish In many of scientific reports such as:
1) The first report of viral infection in Asian sea bass was made by Glazebrook et al. (1990), who described a picorna-like virus associated with mortalities of 15 to 20 d old larvae. This disease was also investigated by Munday et al. (1992) in Asian sea bass and is now recognized as being caused by a piscine nodavirus (Azad et.al., 2005).
2) Also, according to first record of VNN in North America by (Curtis et al., 2001), the viral aetiology of mass mortalities of white seabass, Atractoscion nobilis, cultured in southern California, USA was examined. Disease outbreaks occurred in juvenile fish reared at two culture facilities from June to December 1999, with clinical signs such as anorexia and erratic swimming motion. Microscopic lesions observed in moribund fish included marked vacuolation of brain, spinal cord and retina. The piscine nodavirus (Betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues by electron microscopy, indirect fluorescent antibody test (IFAT), reverse transcription–polymerase chain reaction (RT–PCR), and isolation in cell culture. The agent was identified as one of the four known genotypes of piscine nodavirus. In addition, a similar nodavirus was also detected in fish samples from disease outbreaks at the same facility in 1992. In the last decade, VNN has been reported among cultured populations of marine fish worldwide and this paper is the first record of the agent in North America (Curtis et al., 2001).
Meanwhile, we succeed to isolate and identify of VNN virus as first time in Golden grey mullet (Liza auratus) in the Caspian Sea in 2004 (as attached article).
Furthermore, according to knowledge of my dear friend, Prof. G. Bovo from OIE reference Lab. of VNN, he showed me in EAFP 2013 Conference in Split, Croatia a document about first report of VNN in Senegal in 1996 (In second attached file).
What do you think about original and correct time about first outbreak or occurrence of VNN in the world?
Thanks again
The trypsin can change the VERO receptors used by dengue virus infection.
The 8ug/ml polybrene works well for the viral infection of multiple lung cancer cells in my hands. But recently I got a cell line isolated from mouse lung. 8ug/ml polybrene (with or without virus) can kill all the cells in 24 hours. Has anyone try to infect cells without polybrene? How low will the efficiency be?
viral infection (HIV-HBV-HCV) in hemodialysis patients are common now the question is : is there any viral particles passage possibility from the pores of dialyser ?
During my preparation for state boards in Germany, I have come across a trend particularly for viral infections. In complete blood count analysis during an acute infection, one often finds an increase in Lymphocytes as well as a decrease in Thrombocyte counts. Is the thrombocytopenia a reaction due to this lymphocytopenia (due to bone marrow proliferation and cytokine release), or are the thrombocytes being degraded as a result of the infection (increase in spleen size due to sequestration or increased turnover of thrombocytes)? Any insight on this would be valuable.
Thanx
I have been trying for quite sometimes now, however haven't been able to successfully get the transgene into the cells.
I have tried to use Polybrene 8ug/ml with various MOI starting from 50 up to 1000. I have also used LentiMag (magnetic particle) which suppose to enhance efficiency but still no luck.
I wanted the cells to overexpress a particular transcription factor in one cell, and shRNA specific to the same TF for Knockdown.
I produced the lentivirus based on Didier Trono protocol with PMD2G and PSPAX2 as the packaging and envelope. My transfer vector is purchased from system bio, Cumate inducible lentivirus backbone and the TRIPZ from GE Healthcare.
I used DMEM with 10% FBS as culture media, then collect supernatant over 72 hours and concentrate with the 10kDA CENTRICON.
I then centrifuge it at 10,000g for 4 hours with a sucrose cushion. Pellet is then resuspended with 200ul of PBS. I tested the titre of my virus by infecting 293T with different virus quantity.
I always get approximately 1x10^8 - 1x10^9 IU/ml.
So I don't really know why my human iPSC are not infected properly.
Following are brief protocol on how I carried out the infection:
- I seeded approximately 75,000 cells into 24 well plate a day prior to infection
- On the day of infection I add 8ug/ml of Polybrene to E8 culture media and incubate for 15min
- I then add the appropriate ammount of virus from MOI 50 -1000, incubate at 37oC for 24 hour
- I have also tried double infection the next day however it doesnt seem to improve.
I was wandering if serum from the FBS cause any issue with the low infection rate in my human iPSC??
I would highly appreciate it if anyone could help me :)
- Bacterial and Viral lung infections are a major cause of morbidity and mortality in hospitals as well as community. With neutropenic patients on a raise ..some fungal infections are also increasingly observed.
- If we can have a/or some biomarkers to differentiate bacterial compared to viral and or fungal infections it will be useful in addressing the problem on early stage itself.
- Are there any biomarkers available to differentiate bacterial infections from viral as well as from fungal and or parasitic infections that are available and in use or in research??
Can someone please tell how many " cells" is to be seeded per well for HaCaT cells in a 6 well plate.
How much should be the ideal confluency for lentivirus infection?
Thanks
Apoorva
Is there any relation established between viral infection and pain modulation?
correction to my last entry Should we be testing tissues from GBS patients for Zika?
(Last entry - From what tissues in the body can you isolate Zika virus? - I would like to know whether a real try has been made to isolate the virus from the affected babies, or from infecting mothers and a,Lao if it can be isolated those with GBS should be tested).