Serum - Science topic

Dimas Arya Abdillah , hello.

The antibodies you used for immunoprecipitation (rabbit serum containing the target antibody) and the antibodies used for immunoblotting (HRP-labeled anti-rabbit IgG) are of the same species, so the signals of the heavy and light chains will be strong and signal interference will occur.

So it is recommended to choose a secondary antibody of a different species than the antibody used in the IP experiment for the WB experiment.

If the interference signal is not reduced, you can only perform the WB experiment with the secondary antibody that is species-specific for the detection antibody.

Thank you Kayode Adeyemi for your answer.

We add 1x Laemmly buffer to the beads after the final washing step. Samples are then boiled 5', spun down. Supernatant is pipetted to the SDS-PAGE. Hence, no salt- or acidic elution.

Good point to try w/ non-POI specific IgG :)

ECG findings and Trop I correlation is time domain . Serial ECG is recommended to detect hidden Ischemia

Thank you Mohamed Elseweidy

This can not be explained other than by stating the matrix effect, in my opinion, if all the reagents are healthy and the used procedure is valid.

1)Antibody conjugations are being suppressed by a matrix component, and excess dilutions allow your antibody to bind to its antigen.

2) If dilution is not an option for your workflow, to confirm the hypothesis and to complete the experiment, you may precipitate the targets, cleanse the pellet, and perform the procedure after resuspending.

3) In addition, please inspect your capture and detection antibodies/reagents'...Quality, freshness, dilutions, etc...

Hi Sudarshan, transfection reagents all have certain cytotoxicity. Adjustments can be made from aspects such as the dosage of the reagent, the incubation time, and the cell status.

Feel free to reach out if you have other questions. Hope this helpful!

The only ways I know to get HDL is to infuse the serum with either heparin manganese or phosphotungistic acid. I imagine that electrophoresis will not give a high enough yield.

Thanks Ioannis, I'm glad to hear the kit works for serum too. I'm also happy to hear I'm not the only one running out of wash 1.. I'll keep an eye on eBay :')

Hi Jiexun!

During my graduate studies, when conducting this experiment, we used bovine serum.For more details, you may also refer to the relevant sources.

Hope this helpful. For more experimental details or usage information, feel free to follow me and reach out via private message or comment.

Hello Malcolm Nobre

Thank you for the answer, super appreciated! I'll be looking into it.

As for the ELISA kit, how would you rate it for regular estrogen level measurements, especially in terms of relative levels? Do you think it's good enough or would it still be better to go for LC-MS/MS?

Best,

Hi! I am thinking about the same model. Did you find any solutions?

Hi! Attached you will find some of these data.

Hello @Martina Lorenzati,

At the end I used the protocol by Lonza company and it has been working great, it's the following:

80% standard astrocytes growth media+10%DMSO+10%FBS

Hope this helps,

Mahsa

Thank you so much for directing me to the protocol on ResearchGate. I really appreciate your help! Nicolas Poirier

I used Kia gene auton extraction kit to extract mRNA for gene expression. It is very good.

For specific details just contact Qiagene https://www.qiagen.com/us/contact-us

To quantify ceruloplasmin in serum using SDS-PAGE, prepare serum samples, separate proteins on an SDS-PAGE gel, and stain the gel to visualize ceruloplasmin bands. Use densitometry to measure band intensity and compare it to a standard curve for quantification.

Hello Noha Alziny

Minimum dilution of serum sample is 4x.

You may want to refer to the kit inserts of two companies other than E-Lab science.

Best.

Thank you so much

Dear Dr. Camilla S.A. Davan Wetton

The source of calf serum is from calves aged between 20 days and 12 months. This type of serum contains lower levels of growth factor than FBS, making it less potent for supporting cell proliferation. So, calf serum is used in those cell culture applications where the high growth factor content of FBS is not necessary.

As NIH-3T3 cells have a doubling time of around 18-20 hours, it is highly proliferative. Therefore, calf serum is preferred to FBS as the supplement for the media.

Yes, it does. NIH-3T3 cells are immortalized, but they will still slowly die/ undergo senescence after many passages. So, whenever you use NIH-3T3 cells for any cell-based assay, you should try to use cells of lower passage (preferably less than P-20).

Regards,

Malcolm Nobre

@ Swagata Sarkar- Thankful.

Paul Rutland Thank you for sharing your information, Sir

Gel must be prepared with gelatin and samples applied on this gel.

Physiol. Res. 72 (Suppl. 5): S593-S596, 2023 https://doi.org/10.33549/physiolres.935228

If your Saos-2 cells are attaching but not attaining their typical morphology, there could be a few potential reasons. Here are some suggestions to troubleshoot:

Carefully monitoring and adjusting these conditions can help improve the attachment and morphology of Saos-2 cells. If the issue persists, it might be worth rechecking your protocol or seeking insights from colleagues experienced with this cell line.

Hello Saeedeh Salehi

Both methods are possible. In both cases, it is important to wash the cells to get rid of DMSO (cryoprotectant) which can interfere with RNA extraction. DMSO can slightly but significantly affect RNA structure.

If you plan to extract RNA immediately after thawing, then you may follow the protocol provided below.

1. Thaw the frozen human T cells in less than 2 mins in a 37°C water bath.

2. The thawed cell suspension may be quickly transferred to 10 mL chilled RPMI 1640 medium and centrifuged at room temperature at 400 × g for 10 minutes.

3. The supernatant may be removed, and the cells may be washed once with 10 mL of RPMI 1640 at room temperature and then centrifuged at 400 × g for 10 minutes, also at room temperature.

4. The supernatant may be completely removed, and cells may be subsequently lysed in TRIzol reagent.

I would prefer to culture these cells for 1-2 passages in complete media supplemented with 100 IU of IL-2 and then proceed with RNA extraction because cultured cells provide a more homogeneous population of cells. Moreover, cultured cells will have a faster growth rate, which is linked to protein synthesis. This results in more ribosomes in cultured cells which means more total RNA. After harvesting the cells, perform RNA isolation as soon as possible under cold conditions.

Best.

HAC15 cells will adapt to most sera, but their response to Angiotensin II and potassium changes significantly. Be aware that steroid production in HAC15 cells depends on the applied serum and HAC15 cells cultured in FBS produce other steroids than in CCS. For high aldosterone secretion, I recommend 2.5% UltroSerG serum. HAC15 cells cultured in FBS will divide quicker than in CCS.

There are kits for albumin removal from serum samples. Here is one:

Ismael Aguirre-Maclennan

It seems like you're using a different antibody to those that I've been using. My fixation times were 10 minutes, my cultures are only a few cell layers thick, so they don't require a long fixation time!

What did you use for spiking? Was concentration comparable to the concentration of the high standards?

https://patents.google.com/patent/US20200392461A1/en i think they do it here

This is a slightly complex question.

Essentially you are correct. The cells are growing at levels which would be thought of as castrate in humans. It is more of a question of the 'norms' used for growing these cells lines.

The cells in culture will have adapted their growth to the level they have since they were originally derived, and 10% serum is the recommended growth medium from ATCC, even though it may be lower than human levels. These cells may be considered as being already somewhat androgen independent or adapted compared to normal tissue.

Androgen levels can have a range of concentrations in humans and mammals, and animal sera will also differ - so its important to batch test the serum. Also human patients will have very different levels dependent on being in puberty or elderly - so there is no ideal level - just a 'normal' range. The main thing is to keep it constant for your experiments, and to keep it in line with all other research. It would be impossible and / or very expensive to grow them in pure human serum. Such cells may actually stop growing or die if they were suddenly given the full maximum human level of androgens.

Using charcoal stripped serum will help in measuring androgen - anti-androgen interactions in a controlled system where you can remove all androgens and then add the compounds in a controlled manner, and measure an output - growth or PSA levels are good.

Remember that stripping serum with activated charcoal will remove all small lipophilic compounds - including estrogens, androgens, adrenal androgens, corticosteroids etc. So you may want to take that into account.

Julie Ann Dougherty Thank you!

for small volume of samples, using protein L conjugated beads for immunopreciptation is the best way to remove Igs from the blood samples. after IP, take the top for your western blot..

Hello Pallab Majumder

Yes, SIM-A9 microglia cells can survive w/o serum at least for 24 hours. What is the concentration of amyloid-beta used to stimulate SIM-A9 microglia cells, and the treatment period?

Try treating the cells with 10-20μM Aβf for 24 hours in serum-free DMEM/F-12.

You may want to refer to the protocol in the papers attached below.

Best.

Possibly there is some lysis with a fine needle so do not apply too much suction and take the sample slowly. Check that you are not centrifuging at too high a speed and crushing the red cells....possibly lower g forcr and longer spin. If your animal house has trained technicians taking blood then speak to them about how they do it

Md Ridwan,

I think KI should work as Nal replacement, but it might behave slightly differently, so I would keep an eye on how it affects the results, I shall for sure check for any potassium salts deposits in the solution before proceeding with it. The replacement of sodium lauroyl sulfate should still work but just be mindful, that it can be a bit stronger or much aggressive for cell lysis or protein denaturing, so I would just adjust the concentration accordingly. Without any glycogen, the DNA quantity might be dropping but you should still be fine.

I shall use Tris-HCL at pH8, since the protocol specifies that the extraction solution is maintained at pH8, which is said to be adjusted by Tris-HCl at pH 8. They might be suggesting this pH throughout the process for better results.

If possible, just test the adjusted formulation on a small scale, with minimal serum sample to see how well it works and how good the DNA quantity would be.

Have you considered pre-loading with something like Apixaban?

Hello all,

I found this interesting paper in which authors described their protocol to get rid of the background in their staining.

Hope it can be helpful!

Yes I’m interested because I developed a theory on mental illness based on systemic nitric oxide alterations. It could be interesting to give your compound to psychistric patients to see you f their condition improves thus confirming my theory. Let me know

Isoforms most likely. You also see it when you go from protein to gene or vice versa. You also have the complication of different species.

Here is a publication on this topic Ekta:

Lawlor K, Nazarian A, Lacomis L, Tempst P, Villanueva J. Pathway-based biomarker search by high-throughput proteomics profiling of secretomes. J Proteome Res. 2009 Mar;8(3):1489-503. doi: 10.1021/pr8008572. PMID: 19199430.

Could you post a picture of your cultures at 4 days? Does the % of differentiated cells increase after 4 days?

@Judit Thank you 😊

Dear Aldo

You can probably do this by lowering the pH using an appropriate buffer over a short period of time, or by a competitive approach via adding recombinant proteins similar to the surface proteins of the virus.

Totally avoid reducing agents like 2-mercaptoethanol or dithiothreitol. Following SDS-PAGE under NON-reducing conditions, (heat for only a few min), IgG will land at 150 kDa far away from your target molecule.

Procure a protein A or G ligand attached resin and apply it to plasma. You do not need chromatography. Either batch mode, gravity flow, magnetic bead assisted, or spin column format can bind IgGs and wash out your targets. Immunodepletion would be the most effective approach and can be performed w/o chromatography. Conventional chemical precipitation or physical isolation methods will cause your proteins to be lost. Depending on your choice of analytical platform, protocols can be further discussed.

hi, did you find a solution? I'm also going to try to grow RWPE on RPMI

I would also recommend the albumin and IgG depletion, it will help with the identification of lower abundance proteins.

ELISA determines the quantity of a protein, not the activity. The normal way to quantify the amount by ELISA is to prepare a standard curve, whereby several known amounts of the target protein are tested in the ELISA. In this case, that would be done by adding to the plate various solutions of purified beta-galactoside containing known concentrations of the protein.

It's possible that the mIU/mL readout of your ELISA refers to the quantity of enzyme rather than its activity. The International Unit (IU) is a vague term, and it need not be the same as an enzyme activity unit. You need to find out what an IU of beta-galactosidase means. If it does refer to activity, the conversion to pg/mL would requiring knowing the specific activity (µmol product formed/minute/mg protein) of the particular enzyme-containing sample tested in the ELISA. This would have to be measured with the enzyme activity assay used to define the International Unit. Without that information, the conversion is impossible. Of course, if you perform the enzyme activity assay, what was the purpose of the ELISA?

What is the most accurate technique for measuring the effect of a drug on enzymes? Angelo Mathes

1:2 dilution may do. However, this is dependent on the total volume of sample required for the ELISA. You may find yourself using either 50 or 100ul of serum or plasma.

Hello. Our lab has found that a good dilution factor for VCAM-1 within both postmortem and antemortem human serum is 50X. I hope this helps.

The assay dictates which tube to use. EDTA and sodium citrate containers contain preservatives that interfere with clinical chemistry assays. Red top clotted serum tubes can be used for any assay a yellow top serum gel tube is used. The only difference is the red top tubes don't contain gel to separate the cells from the serum.

There are tube guides published on the internet.

Additionally, with lithium heparin tubes there is noworry about clot formation (normally) and this is a time saver in getting the blood analyzed.

Hi Wang and Camila, could you solve the problem??

I would like to buy K/BxN serum but where can I do?

Dear Manuele Martinelli

Thanks very much for your reply - I will definitely look into this!

Best wishes,

Victoria

Thanks Malcolm Nobre.

Differentiation is definitely my next way out. But I still have some experiments to be carried out with undifferentiated cells. I have seen adding extra serum, 11% total helps the cells spread out of the clumps.

You should first try to find out whether your secondary antibody alone (without applying the primary antibody) gives the same result > negative control; if so, use higher antibody dilutions and titer it until the background is minimal. Then apply the primary antibody and the secondary antibody in the optimal dilution you determined in the previous step.

Another option is to prolong the incubation with blocking buffer or to try different blocking solutions, e.g. BSA.

The impact of different blood collection methods, such as intravenous catheterization versus venipuncture, on the potassium concentration and hemolysis index in serum and heparin plasma samples are observed. Comprehending these distinctions is essential for evaluating sample quality and guaranteeing dependable laboratory testing outcomes. When interpreting hemolysis and potassium levels, laboratories should take the technique of blood collection into account. Appropriate quality control procedures should be put in place to minimize pre-analytical impacts and preserve the integrity of test results.

A number of things, including thawed samples, cracked vials, improperly sealed vials, empty vials, and severe hemolysis, can lead to unsatisfactory specimens. Inadequate specimens are recorded on the transmittal using the sample condition codes when they happen infrequently.

Reference. Kwiterovich, Peter. Laboratory Procedure Manual of Total Choloesterol.

According to Farhana and Lappin (2023), serum is typically considered a superior sample to plasma for lactate dehydrogenase (LDH) testing. This preference comes from the fact that serum, which is acquired by letting blood clot and then extracting the clot, lessens the possibility of interference from cells generating LDH during processing samples. On the other hand, hemolysis can occur in anticoagulant-collected plasma, leading to erroneously high LDH values. The serum reduces errors related to cell breakdown, hence ensuring a more accurate and consistent assessment of LDH activity.

Farhana, A., & Lappin, S. L. (2023, May 1). Biochemistry, lactate dehydrogenase. StatPearls - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK557536/

Beyond plasma and serum, several other sample variations could be explored to improve turnaround time (TAT) and sample quality in clinical or research settings. Here are a few possibilities:

1. Urine: Urine samples are readily obtainable and contain valuable diagnostic information. Further exploration could lead to quicker and more effective testing methods for a range of conditions.

2. Saliva: Saliva samples are easily accessible and offer diverse biomarkers for diagnostics. Enhancing our understanding and handling of saliva could streamline testing processes, improving accuracy and efficiency.

3. CSF: CSF samples hold critical insights into neurological conditions. Advancements in CSF sample processing and analysis techniques could enhance diagnostic speed and reliability for brain-related issues.

4. Stool: Stool samples provide significant insights into gastrointestinal health. Research into improved handling and analysis methods could optimize diagnostic accuracy and turnaround time for gut-related conditions.

5. Sweat: Sweat contains diagnostic markers for various conditions. Investigating enhanced methods for sweat sample collection and analysis may expedite and refine diagnostic processes.

6. Tissue: Tissue samples play a crucial role in diagnosing organ-specific diseases. Refining techniques for tissue sample processing and analysis could lead to swifter and more precise disease diagnosis.

Hello Caitlin! According to the U.S. Department of Health and Human Services (2009), here are the following specimen considerations:

References:

U.S. Department of Health and Human Services. (2009). Biosafety in Microbiological and Biomedical Laboratories (5th ed.) [E-book]. HHS Publication No. (CDC) 21-1112. https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

The factors to be considered in order to avoid discrepancies in values for LDH measurement include variation in tube brands, plasma contamination with platelets, fragmentation of platelets, and fragmentation of erythrocytes. Platelet residues and lysis in plasma, as well as hemolysis and coagulation in serum, can also have an influence on LDH measurement. Overall, the type of sample, preparation process, and tube selection must all be considered in order to obtain an accurate evaluation and interpretation for your LDH measurement.

Lithium heparinized plasma (LIH) tubes can improve the sample quality, reduce aspiration errors, and decrease the turnaround time (TAT). In the study before switching to plasma, serum samples had 0.88% aspiration errors, but after switching to plasma, they had 0.44%. Aspiration errors are statistically significant, as they're one of the factors that can influence TAT. The median values from sample acceptance to device entry decreased from 21 to 17 minutes after switching to plasma samples, thus improving the TAT. These factors open up the possibility for improved quality healthcare and reduce the workload of hospital staff.

Lithium heparin gel tubes are now used in the laboratory due to several advantages over other types of blood collection tubes. In order to avoid clotting or thrombin activation, which in turn stops the synthesis of fibrin from fibrinogen, heparin stimulates the inhibition of thrombin and Factor X. It is advised to use lithium heparin since it is the least likely to affect the outcomes of tests for other ions, such as sodium. Transitioning from gel separatory serum tubes to lithium heparin gel tubes can enhance clinical laboratory efficiency and accuracy by reducing processing time, minimizing sample interference, improving sample quality, and expanding the range of tests that can be performed reliably. However, proper training and adaptation to new protocols are crucial to fully realize these benefits.

The preservation of enzyme activity is the main connection between the anticoagulants used in blood collection tubes and the lactate dehydrogenase (LDH) activity in plasma or serum samples. Cells contain an enzyme called LDH, whose activity can be evaluated in serum or plasma samples to determine whether a disease or tissue damage has occurred.

There are previous studies that show the same result to the TAT. Some studies such as the study of Hetu et al. and Ramakers et al. For Hetu et al.it studied Potassium test and concluded a significant decrease in the average time in terms of sample reception and result confirmation. This is similar to Ramakers et al. wherein there is a decreased median TAT with the use of Barricor tubes. Another study is from Badiou et al. that reported the same result using Barricor tubes instead of gel LIH tubes. In this study, the TAT was reduced due to the use of plasma instead of serum that needs to be clotted which is the factor that increases the TAT.

Oguzhan Zhengi utilized the Clinical and Laboratory Standards Institute (CLSI) Criteria - GP34-A:2010 upon handling his specimens for testing. It is a consensus-based medical laboratory standards which is most widely recognized resource for continually improving testing quality, safety, and efficiency. In the Methodology part of the journal, he stated that recommendations of the tube manufacturers were followed concerning tube inversions, clotting time, and centrifugation characteristics while still adhering to the said CLSI criteria. The CLSI Criteria utilized contains specific guidelines on the validation and verification of tubes for venous and capillary blood specimen collections. Through this, the samples were centrifuged for 1800 g for 10 minutes and visual inspection of sample quality indicators which included hemolysis, lipemia, icterus, fibrin, and clots were taken into careful consideration.

Most tests showed no significant difference between the serum and LIH tubes. For some analytes, total error (TE) values exceeded the total allowable error (TEa) limits. Insulin TE value did not exceed TEa but consumed nearly all its error budget. Plasma tubes could be used instead of serum tubes for most tests, except for lactate dehydrogenase (LDH). Plasma tubes improved sample quality, reduced the incidence of aspiration errors, and decreased TAT in the emergency laboratory.

In order to assess the comparability of blood tubes and LIH tubes for clinical chemistry and immunoassay testing, this question explores the interpretation and consequences of statistical studies, such as Bland-Altman plots and regression analysis. Additionally, it is a graphical representation of the difference between two variables, such as the effectiveness of lithium heparinized plasma and serum in clinical chemistry and immunoassays. Through the clarification of statistical subtleties and the identification of noteworthy discoveries, scholars and researchers can enhance our comprehension of the fundamental mechanisms propelling distinctions—or absence thereof—between the two varieties of tubes. The scientific community, clinicians, and laboratory staff who choose tests and interpret results can all benefit from this research. Regression analysis and Bland-Altman plots, however, demonstrated that the majority of tests did not demonstrate any appreciable variations between serum and LIH. Nevertheless, the biological variation (BV) database's tolerance for error restrictions (TEa) was surpassed by a few analytes in this specific journal.

Underfilling of blood collection tubes occurs during the pre-analytical phase. However, this phase of testing is considered the most vulnerable part of the total testing process and poses the greatest challenges to laboratory professionals (Simundic & Lippi, 2012). It is also regarded as less standardized and a significant contributor to laboratory errors (Neuwinger et al., 2019). Therefore, continuous education, certification, and training of healthcare practitioners in blood drawing responsibilities, along with improved standardization of phlebotomy techniques and dissemination of operative guidelines, would enhance the chance of obtaining specimens of consistent quality (Lippi et al., 2006).

I would suggest using mouse anti-CD3 and CD19 labeled with fluorescence (FL1) for B and T cells, while using a anti-human Fc labeled with a different FL2 to evaluate the pre-exist anti-lymphocyte antibody. It will be a consecutive gating of first gate is SSC/FSC, second gate is FL1/FL2, while double positive cells are what you want.

Best

Underfilled vacuum blood collection tubes can potentially affect the isoenzyme level of lactate dehydrogenase (LDH) due to several reasons:

Hemolysis: Underfilled tubes may increase the risk of hemolysis during blood collection or transportation. Hemolysis, the rupture of red blood cells, can release cellular contents, including LDH, into the plasma or serum. This release can artificially elevate LDH levels, affecting the accuracy of LDH isoenzyme measurements.

Sample Dilution: Underfilled tubes may contain less anticoagulant or clot activator, leading to insufficient blood volume for proper mixing. Inadequate mixing can result in sample dilution or improper clotting, potentially affecting the accuracy of LDH measurements and isoenzyme analysis.

Incomplete Fill: Underfilled tubes may not provide sufficient blood volume for proper sample analysis, particularly in automated laboratory systems. Incomplete filling can lead to inaccurate measurements or incomplete testing, affecting the reliability of LDH isoenzyme results.

Clot Formation: Inadequate filling of blood collection tubes may result in improper clot formation or partial clotting. Clots can trap cellular components, including LDH, affecting the distribution of LDH isoenzymes in the sample and potentially leading to erroneous results.

As mentioned by the authors of a journal titled “The transition from gel separatory serum to lithium heparin gel tubes in the clinical laboratory” the reason for a negative bias for LDH was not fully understood. However, it is believed that LDH may be released from platelets and other cells into plasma during centrifugation. However, as claimed by Peake, M. J., Pejakovic, M., Alderman, M. J., Penberthy, L. A., and Walmsley, R. N., in a journal article titled “Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma,” platelets are said to impede lactate dehydrogenase activity in plasma under conditions of low osmolality. However, their observations challenge these claims, suggesting that what appears as inhibition may actually stem from optical interference by platelets during the reaction.

Their investigation indicates that when platelet lysis is prevented and optical interference is corrected for, both platelet-rich plasma and platelet-poor plasma, as well as serum, exhibit similar levels of lactate dehydrogenase activity. Additionally, it’s worth noting that platelet contamination can lead to unexpected issues when assessing lactate dehydrogenase with centrifugal analyzers. As mentioned in the journal article, the results may vary significantly depending on the volume of diluent added with the sample, potentially causing both higher and lower readings, introducing considerable within-run fluctuations in activity.

Furthermore, platelets can scatter light during the LDH assay, leading to optical interference that affects the accuracy of the measurement. This interference can result in falsely elevated or depressed LDH levels, depending on the extent of platelet contamination and the specific assay method used. In addition, platelets contain LDH enzymes, which can contribute to the overall LDH activity measured in a plasma specimen. If platelets are not adequately removed or lysed during sample processing, their LDH activity may artificially inflate the total LDH measurement, leading to inaccurate results.

Reference: Peake MJ, Pejakovic M, Alderman MJ, Penberthy LA, Walmsley RN. Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma. Clin Chem. 1984 Apr;30(4):518-20. PMID: 6705193.

The Warburg Effect characterizes the metabolic shift in cancer cells, prioritizing increased glucose uptake and lactate fermentation despite functional mitochondria, to fuel growth and survival (Liberti et. al., 2016). Glucose metabolism generates ATP crucial for cellular energy needs and yields lactate or CO2 as end products depending on full mitochondrial oxidation or fermentation. Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, regenerating NADH to NAD+ (Alegre et al., 2015). Moreover, in tumors, increased glucose uptake leads to elevated lactate production. In conclusion, the Warburg Effect does not directly increase LDH levels, instead, LDH activity is central to the metabolic rewiring observed in cancer cells undergoing the Warburg Effect resulting in increased levels of LDH in serum and plasma.

References:

Liberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016 Mar;41(3):211-218. doi: 10.1016/j.tibs.2015.12.001. Epub 2016 Jan 5. Erratum in: Trends Biochem Sci. 2016 Mar;41(3):287. Erratum in: Trends Biochem Sci. 2016 Mar;41(3):287. PMID: 26778478; PMCID: PMC4783224.

Alegre, E., Sammamed, M., Fernández-Landázuri, S., Zubiri, L., & González, Á. (2015). Circulating biomarkers in malignant melanoma. In Advances in clinical chemistry (pp. 47–89). https://doi.org/10.1016/bs.acc.2014.12.002

Hi Keller, Thanks for your reply!!

You can use EasyPure® Genomic DNA Kit, 200ul from clotted blood is enough to start your protocol then follow the pamphlet protocol

You're correct that Ab often perform quite differently in Western versus ELISA. But it's something you can try.

What makes you think your antiserum works at all? if you have any titer measurement you can use that to estimate a working dilution for IP.

Or you could always just guess. A dilution of 1:1000 should work, if the antiserum is reasonably potent. You may be able to go down to 1/10k or 1/100k.

Follow your local rules for use of human tissue in lab research. I recommend you always heat treat human serum to kill viruses, and then still treat the reagent as potentially infectious. My niece caught HepB from handling a human serum sample is a hospital lab.

Hello Subhadip Kundu

I feel these normal fibroblasts are getting activated during invitro propagation. There is a possibility that the culture conditions may be contributing to the activation of these normal fibroblasts. These fibroblasts can get activated in response to various stimuli present in the culture environment. One such stimuli being the growth factors present in the culture media or some kind of stress experienced by the cells in culture.

So, try to find the source and adopt measures to minimize the activation of these cells.

Best.