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Platelets - Science topic

Scientific research concerning platelets.
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what is the normal range of clopidogrel sensitivity and resistance ? and what is the range of platelet function P2Y12 test and platelet function base test for clopidogrel sensitivity and resistance ?
for instance : 60 year old male patient with platelet function P2Y12 test ( 54 ) and platelet function base ( 194) .
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I suppose you mean `high on-treatment platelet reactivity'. Several tests are used for diagnosis, thus the definition of 'normal values' and the cut-off for positive diagnosis depend on assay used. Generally, the 50% or more inhibition of platelet reactivity is considered as 'adequate' response to clopidogrel.
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Hello,
I using human serum and platelet lysates as growth supplements for the cell culture of monocytes and I am now looking for animal-free alternatives that will prevent coagulation. Ideally I would like to find synthetic heparin but any other anti-coagulant could be worth to try as well.
Please let me know if you have any good recommendations!
All best,
Alexandra
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Good to know! Thank you for all the information.
All best,
Alexandra
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Immune thrombocytopenic purpura (ITP) is a blood disorder characterized by a decrease in the number of platelets in the blood. Platelets are cells in the blood that help stop bleeding. A decrease in platelets can cause easy bruising, bleeding gums, and internal bleeding. This disease is caused by an immune reaction against one's own platelets. It has also been called autoimmune thrombocytopenic purpura.
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I am trying to prepare platelets (from fresh blood) for flow cytometry. After getting a platelet pellet from fresh plasma, I resuspend it in either 1mL or 100µL of tyrodes solution, before incubating it with antibodies and centrifuging to wash. Almost every time, the platelets begin to clump and sometimes the solution itself turns very viscous. This happens when use tyrodes with or without phosphate. When I re-suspend them in PBS instead however (without Ca/Mg), they are fine. I assumed this is because they are getting activated, but adding EGTA does not fix the problem. Any ideas on what is causing this or recommendations to fix it? Thank you!
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I might be a bit late, but could you try activating the platelets, add a fixation step of 15 min in paraformaldehyde (1% final concentration) to prevent platelet activation during further steps and then add Ca2+ to allow annexin V labeling?
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in resource limited settings, the may be need to carry out platelet function test without the use of equipment. which is the best technique and is the result comparable to standard platelet function test using automated techniques?
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Functional platelet studies after a period of high interest are currently less frequent because attempts to correlate in-vitro outcomes with the in-vivo platelet function have not been encouraging. So, thromboelastography and aggregometry with ADP, ristocetin and other agents are practically abandoned. The in vivo studies, such as the bleeding time or the vascular fragility test, are burdened by the lack of standardization and, in any case, they are global tests that investigate different phases of the platelet vascular response. Ultimately, only generic automated tests such as total and reticulated platelet counts provide partial information that is not always easily interpretable.
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Would the following results for CBC WITH DIFFERENTIAL be representative of a pregnant or non pregnant female. If pregnant, why? And approximately what gestation period.
WBC 10.7 K/cumm
RBC 4.01 M/cumm
Hemoglobin 12.8 g/dL
Hematocrit 37.7%
MCV 94.0 fL
MCH 31.9 pg
MCHC 34.0 g/dL
RDW 13.2
Platelets 309 K/cumm
Mean Platelet Volume 9.2 fL
Platelets Distribution Width 9.4 fL
Neutrophil % 69%
Neut. Absolute 7.4 K/cumm
Lymphocyte % 23 %
Lymph. Absolute 2.5 K/cumm
Monocyte %. 6%
Mono. Absolute 0.6 K/cumm
Eosinophil % 2%
Eos. Absolute 0.2 K/cumm
Basophil % 0%
Baso. Absolute 0 K/cumm
additional
BUN/CREATININE/LYTES - Details
BUN 13 mg/dL
Creatinine 0.7 mg/dL
Sodium 139 mEq/L
Potassium 4.3 mEq/L
Chloride 105 mEq/L
Carbon Dioxide 25 mEq/L
Thank you
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I think it carries very little sensitivity.
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I have recently been conducting experiments on platelet aggregation in rats using optical turbidimetry. Use ADP as a platelet activator. However, the aggregation rate is only 20 to 30 percent. I tried different concentrations and inducers, but they didn't make much difference.
Here's how I did it
1、Blood was collected from rat abdominal aorta by vacuum vasculature and sodium citrate was anticoagulant. Then centrifuge at 260g for 10min
2、Drain the supernatant(PRP) into another clean EP tube and let stand at room temperature for 30min
3、The remaining blood was centrifuged at 3200rpm for 10min to obtain PPP
4、Set the transmittance of PPP to 100%. Add 300μ PRP into a colorimetric cup, preheat it at 37℃ for 1 min, then add ADP (10μM), stir continuously, and measure the change of absorbance within 5 min
Despite looking at the literature, I still can't figure out what the problem is. Thanks for your advice in advance.
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Dear Fang Yafei!
You should add calcium chloride or calcium gluconate. It can resolve your problem.
Sincerely yours,
Artem
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I‘m interested to study the percentage of CD8:platelet aggregate in whole blood of patients collected in sodium citrate tube. Reason is that we want to study ‘real time’ interactions in the blood without disturbing them too much.
I’ve read papers online and the methods they use are either
1) isolating platelets and coculture with platelets or
2) direct staining in whole blood then lyse RBC.
My question is
1) is RBC lysis necessary (using lysing solution, no wash)? Can 0.2%PFA lyse RBC?
2) Do you use Fc block to block other interaction?
3) Please share protocols that would be useful
Thanks!
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Not sure to fully capture the question about the Fc fragment but it may bind to many cells - from lymphocytes, basophils, ..., platelets. So you may have non specific or false positive signals when performing your flow cytometry. Best is to use blocking reagents (IgG, anti-Fc receptor antibodies) to saturate the receptors before staining your cells with labeled antibodies.
Red blood cells should be eliminated. Lysis would help to clear some confounding factors and to sort your flow cytometry deliverables.
Most of the time : Ice-cold ammonium chloride lysis buffer : NH4Cl, NaHCO3, EDTA, and washed several times along the centrifugation with PBS.
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How can I get rid of abundant protein (albumin, immunoglobulins) from exosomes isolated from platelets after ultracentrifugation? I need a simple, quick, and cheap solution.
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There are gels to remove albumine from serum samples for proteonics studies, check whether these can be used for exosome preparations. Alternatively, spin the exosomes through a sucrose cushion, soluble proteins should stay in the sample zone. With metrizamide such cushions can be even isotonic, but it is much more expensive.
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How can I get rid of abundant protein (albumin, immunoglobulins) from exosomes isolated from platelets? Exosomes were isolated from platelets by ultracentrifugation. I need a simple, quick and cheap solution.
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you can wash the platelet in tyrod buffer and then assay the protein content
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I am studying the effect of RFID on the viability of platelet units (Transfusion).
Several units (bags) of platelets will be used in the study. Each unit is likely to be taken from a different donor so the start point for each analytical test to be performed will be different.
The random units will be split into to 2 equal size groups.
One group will be placed, and left for the period of the study, on a platelet agitator that has NO RFID field applied.
The second group will be placed, and left for the period of the study, on a platelet agitator WITH an RFID field applied.
There is no difference in any other way between the agitators.
An analytical test with a numerical result (e.g. pH) will be performed on an aliquot of every unit in the study each day for 8 days (days 1, 2, 3, 4, 5, 6, 7 and 8). The remainder of each unit is placed back on the relevant agitator until the next days aliquot is taken out.
I will be looking to see if there is any statistically significant difference between the 2 groups as an indication of if the RFID field applied has had any effect on the platelets in each group.
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You can see how we did test mice over time:
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The patient's pulse suddenly dropped to 40 ÷ 42 beats per minute, and this for two weeks. Blood pressure did not decrease, and is at the level of 120/140 x 65/80. All biochemical blood analysis parameters are within normal limits, including platelets, AST, ALT, direct bilirubin. Indirect bilirubin - 34 µmol / l. No signs of a heart attack or ischemia were detected. What could it be? Intoxication?
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Dear O. P. Strakhova,
I still believe that Gilbert's syndrome has to be excluded as a causes for the mildly elevated indirect hyperbilrubinaema and bradycardia.
Regards.
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I want to remove platelets from my human plasma samples to run after that qPCR (checking mtDNA/nuclear DNA copy number). Because of the known problem of over quantification if there are platelets...
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You can check it on hemocytometer using Trypan blue dye at 20X objective and look for any round bright dots
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Assessment of platelet function when the blood platelet levels are low
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Platelet transfusion is indicated for patients with clinically significant bleeding in whom thrombocytopenia is thought to be a major contributory factor, even if the platelet count is >10x109/L.
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What is the best way to draw plot ( Graph) to present engraftment data
Which is normally include the time it takes for platelets to reach >20 and neutrophil to reach >0.5 ?
I want to present my data in a conference yet? I am not sure what is the best plot?
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It would be better to use a line graph with markers of days on the abscissa and quantitative indicators of cell composition on the ordinate, on my opinion.
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I work in a cancer lab and have been trying to isolate and quantify platelets without the use of any advanced hematological instruments. I don't need pure platelets, so I've just been generating platelet-rich plasma and trying to get a rough count using a neubauer chamber. With this method though it's incredibly difficult to figure out what I'm looking at under the microscope, and counting platelets has been impossible. Does anyone have experience with this?
Current PRP isolation protocol:
1. Collect blood from mice after CO2 euthanasia via the posterior vena cava, collect into sodium citrate (1:9)
2. Centrifuge whole blood @300g for 20min
3. Collect upper 2/3 plasma fraction
4. Centrifuge fraction @800g for 15min
5. Resuspend in PIPES buffer, pH 7.4
6. Dilute 1:20 and add 10uL to Neubauer chamber
7. Allow cells to settle in moist environment for 15min, try to count
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Dear Ma'am Savannah Free please read these articles, they might be helpful for your study.
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I have red blood cells isolated according to the protocol attached. I would like to know what stain would be required for flow cytometry to identify the red blood cells for gating. Also, if I want to check for contamination by other stuff like platelets or white blood cells, what additional stains would I need?
Thank you!!
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You can identify the RBC quite nicely according to forward and sideward scatter. Platelets are much smaller and you can gate most of the WBC away. I would not necessarily freeze the RBC before flow cytometry. The type of staining you use, depends essentially on what you want to do research on.
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I am a master student from China. I need to seperate platelet-rich plasma and count with a light microscope and a blood count plate. I want to know if any method can help me see the platelet clearlly. Thanks for your answer!
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Above both are correct
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Hello, I am investigating the activation of platelets with the use of flow cytometry. I am specifically looking at their activation when agonists such as ADP, collagen, and thrombin are introduced in whole blood. So far I have been staining the cells first with various markers then agonists are introduced and last step is to fix them with formaldehyde.
I am wondering what would happen if i were to fix the whole blood, then stain and put agonists?
Alternatively I can stain first, then fix the whole blood then put agonists.
Or even put agonists, then fix the whole blood and later on stain?
Can you please give me your thoughts as well as pros and cons on that matter?
Please bear in mind that I am looking at the activation of platelets.
Thank you very much
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Thank you very much! So, if I unterstood well, you are using the whole blood, then stain and fix or fix and stain? I will probably follow fixation after staining. What did you mean by diluting with staining solution?
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I am working with platelets and getting blotches whenever I am running a gel. Why is it so? How can I resolve this problem?
I am attaching my Coomassie gel image as a reference.
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Some authors describe it as an excessive amount of protein in the serum. Take a look at "Morin S et al. A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaciis implicated in the circulative transmission of tomato yellow leaf curl virus. Virology. 1999;256(1):75-84."
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Hello everyone. I have begun on analyzing platelets using a flow cytometry. As I have never done analysis of this cell type, can you please help me out.
For now I am using markers CD61 FITC/BV510, PAC-1 FITC, CD62P PE (10 micro liters each marker), currently ADP (10 micro liters) is going to be used as an activator, and whole blood (5 micro liters).
On my last analysis I used CD14 APC (monocytes), CD61 FITC (platelets) plus whole blood (no ADP) to see a clear population of platelets. This was achieved and a clear population was seen, however once ADP was introduced I was not able to see a clear population of activated platelets.
Should I use less ADP (perhaps 5 micro liters), less incubation (from 10 min to 5 min), increase the staining buffer ?
Thank you in advance
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Completely agree with Waltraud C Schrottmaier comment.
The concentration of stimulant matter a lot. At higher concentration the stimulants become cytotoxic. You must titrate the amount of antibodies in different assays. Most of the antibodies works well in 1:10 to 1:50 dilution of recommended concentration.
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When an anti-coagulated blood sample is taken, how can we calculate the number of RBC, WBC, and platelets using the flow cytometry technique? Do we need to separate the components in the blood sample and then introduce the sample to the flow cytometer?
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Dear Kritika Ramchiary to you first question:
You can count the blood components by flowcytometry just by adding true count beads to the sample while acquiring.
Otherwise you will get the counts but not the concentration (numbers/uL). SO the true counts beads has a defined number in per microlitter volume. you add 10 microlitter beads to sample and take 900 beads/ ul is their defined value. so when your cytometer will count 900 beads in its column, it will consider it 1 ul volume and will give you value of L, M, N in per microlitter.
You can definitely count RBC as well by this method, but you know RBC are seen in Log scale while WBCs are seen on Linear scale in flowcytometer, so getting value of both the cell types need to be optimized. It might be done (does not seems impossible), if you want to do.
To your next question:
Yes RBC and WBCs can be differentiated by their scatter pattern as well as cell surface markers.
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I am in a lab without microscopes. Can anyone recommend an affordable method / product for cell counting of human platelets (~1.5 - 3um)?
Thank you!
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As far as the suggested methodology with hematocrit capillary, numerous unknown variables interfere with the supposed counting, PLT volume in primis. As an example, some thrombocytopenias having an increased MPV could have a falsely higher "counting" with that method
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As soon as washed platelets are prepared at the desired concentration, and left at room temperature (with no apyrase), how long does it take for them to start activating??
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Hi Rh Rj, it depends on the platelet preparation methods that you use. I add PGI2 to prevent platelet aggregation during the centrifugation steps. PGI2 gets deactivated after 30 min. Therefore, I wait for at least 30 min (it is called resting) at RT on my bench, I do not agitate them during resting. Then you can start with the experiments.
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Hello!
Is it possible to activate human platelets while preventing aggregation? Maybe with a low platelet concentration and by keeping the solution in motion while activating and fixing?
My goal is to have stained (with CMFDA dye), activated (ADP?) and fixed (formaldehyde?) single platelets.
Background information on the platelets I will have available:
Human platelets are isolated from platelet rich plasma (PRP). In the isolation process Apyrase and EGTA are added to prevent aggregation. The platelets are washed with CGS-buffer (NaCl, Citrate, Glucose) and finally resuspended in buffer. After this step I will receive the platelets.
Greetings
Matthias
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Your washed platelets shouldn't have any extracellular fibrinogen so you can safely activate them with minimal aggregation. A simple procedure I have used to activate washed platelets and fix them is to:
  1. Add agonist to an empty eppendorff
  2. Add platelets to dilute agonist to working concentration (~30 minutes)
  3. Fix with paraformaldehyde (10 minutes)
  4. Quench excess fixative with 2M tris-glycine solution
  5. Stain samples as you wish
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Hello. I am testing PGE1 on my washed platelets, which is suppose to inhibit platelet activation. If the platelets have been left at room temperature for a while, and possibly activated, in this case will PGE1 not do anything? I am trying to understand why my platelets are not behaving as expected to PGE1.
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Hi Rh Rj . Have you tried treating PRP with PGE1 before washing? Also platelets need constant rocking motion.
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Hello. Does anyone know of any studies where the cells or platelets were incubated for a very short amount of time in PFA and it was sufficient for fixation? For example, 30 seconds or 1 minute in 1.5% PFA, or something similar.
Thank you.
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Dear Rh Rj , We usually keep our cells in 1% PFA for overnight at 4 degree C and treat at 4% PFA for 10 minutes at RT.
In one of our research on Monocyte-Platlet aggregation, we fixed the platlets as we all lysed RBC at once with RBC lysis buffer (BD Biosciences).
Lowering concentration of PFA need to be compensated with increasing time.
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Hello,
I am studying neutrophil-platelet aggregates in whole blood samples for a project. Can I use MPO for neutrophils (we have also CD11b, CD15 and CD16) for neutrophil-platelet aggregates? (Since for MPO I should use intracellular staining, I was wondering if it harms platelets?)
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Dear Basak!
Yes, you can use MPO to study neutrophill-platelet aggregation
Analysis of MPO binding to human platelet membrane
Freshly washed platelets (2×108 cells/ml) were incubated with gentle shaking in a phosphate buffered saline (PBS) (10 mM Na2HPO4/KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), containing 1 mM CaCl2 and 0.5 mM MgCl2 with or without MPO for 10 minutes at 37°C. In some experiments cell suspension was supplemented with sugars (α-methyl-D-mannoside, lactose or N-acetyl-D-glucosamine at the final concentrations of 60 mM) or either with 50% embryonic calf serum or platelet-poor plasma. Then cells were applied on poly-L-lysine-coated glass slides. After 30 minutes the samples were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes, then again washed with PBS. To visualize MPO, cover slips with platelets were incubated for 1 hour in PBS containing anti-MPO antibody (1:5,000 dilution). After being washed with PBS three times to eliminate the excess of antibodies cover slips were kept for 1 hour in PBS containing anti-rat IgG-FITC antibodies (1:10,000 dilution). Then cells were again washed three times with PBS before mounting on slides. Images were acquired using laser scanning confocal microscope LSM 510 META (Carl Zeiss, Germany) with an immersion lens Plan Apochromat 63×/1.4 Oil (Carl Zeiss, Germany) and processed using LSM 510 software.
Measurement of human platelet aggregation
Platelet aggregation studies were performed using both optical and impedance aggregometry. Platelet aggregation was detected by recording changes in light transmission at 540 nm of cell suspensions at 37°C on a computerized aggregometer AP 2110 from SOLAR (Minsk, Belarus). 400 µl PRP (2.5×108 cells/ml) or washed platelets (2.5×108 cells/ml) in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2 were incubated at 37°C with stirring for 3 minutes (with or without MPO) before adding an agonist (ADP to PRP and thrombin to washed platelets). Aggregation curves were recorded for 10 minutes. To quantify agonist-induced aggregation we used the maximal rate of cell aggregation calculated automatically by aggregometer software. Electronic impedance aggregation measurements were performed using an impedance lumi-aggregometer Chrono-log 700 (USA). An aliquot of whole blood (0.5 ml) was diluted with an equal volume of prewarmed isotonic saline and incubated for 5 minutes at 37°C with or without MPO. Impedance of each sample was monitored in sequential 1-minute intervals until a stable baseline was established. Then ADP (5 µM) was added and aggregation was continuously monitored. Impedance aggregometry results are expressed as amplitude (or maximum aggregation) [ohm] at 6 minutes after reagent addition.
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I should have one peak of stained platelets. I am staining an intracellular protein after fixing and permeabilising. I keep getting two peaks in various samples, as if some of the platelets are not being stained. This makes it difficult to analyse data.
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Is it possible that some of the platelets have degranulated prior to fixation? What are you using for fixation and is it fresh? I would also recommend going back to your FSC and SSC plots and seeing if the phenotype segregation can be attributed to differences in size (FSC and /or granularity (SSC)
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Two days ago, we thawed Wharton's jelly human mesenchymal stem cells in the following media composition: DMEM-low glucose + 2 U/mL Heparin + 5% HPL + 1% antibiotic antimycotic + 2% HEPES. Attached is the picture of cells in P5. Today, we changed the media using the complete media that we already prepared two days ago. We took out the media from 4 degree Celsius and warmed it in a water bath. After changing the media, cells morphology changed (please refer "wj after changing media"). This happened after 30 minutes to 1 hour after changing media. Do we need to freshly prepare the media every time we want to use it?
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After viewing the images change of media not suggested
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I would like to label some fixed cells/platelets with CM-H2DCFDA (General Oxidative Stress Indicator) dye . I would like to know if this is possible in fixed cells. Does the dye bind covalently to intracellular targets? Do fixed cells even have ROS?
According to ThermoFisher Scientific:
CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells. This indicator exhibits much better retention in live cells than H2DCFDA. CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facilitating long-term studies.
Thank you.
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CM-H2DCFDA dye was used for analysis the oxidative stress of cancer cells after drug treat. In here we did not fix the cells. Analysis with FCM. In cell cycle analysis cells was fixed with 70% ethanol.
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Good morning all.
Just picking brains here if I can please? I am interested in measuring circulating concentrations of angiopoietin-1 (angpt-1) in patient samples. Several manufacturers indicate that plasma needs to be platelet free for measurement as they release angpt-1 upon degranulation and may contaminate sample. Samples have already been processed in-hospital and I cannot generate the platelet free extract (frozen when I collect). The same manufacturers state, however, that serum is OK for measuring this protein. This seems strange to me, since serum is derived from clotted blood which would involve platelet degranulation? Surely this would involve massive release of platelet products and create further contamination of sample?
Thoughts????
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Thanks all for the responses. No easy answer it seems! I've ordered another set of tests to be run on plasmas that were collected at the same time as the serum to see if there is any difference.
@Jan - will look at the platelet count as you suggested. This is probably relevant to the pathology I'm interested in too!
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am looking for a protocol to extract microRNA from platelets where I can use for qrt-PCR
any recommendations please?
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-- For microRNA work, go for Qiagen products, their all microRNA research products are the best in the market.
-- You can isolate total RNA from your samples using TriZol method. Then, you can use the Qiagen miscript-II-RT kit for cDNA synthesis. This kit will convert all miRNA present in the sample into miRNA-specific cDNA. Now, you can analyze your miRNA of interest by SYBr qRT-PCR using miRNA primers.
-- Even you can buy specific miRNA isolation kit and isolate miRNA. But ultimately cDNA synthesis will matter for further qRT-PCR.
Keep this point in a note that
-> All miRNA cDNA synthesis kit not works with SYBr, many only works with Taqman. So before buying check its requirements.
-> Prefer to use a pre-designed miRNA primer.
-> Use at least 3 endogenous control.
Now, miRNA primer has universal sequences. you will find the stem-loop & mature miRNA sequence in the miRDB database. Well, as I said, prefer predesigned primers of your interested miRNA from any standard company. Nowadays, Companies do a slight modification or say a technology, which enhances stability and avoids unwanted binding. Qiagen has LNA tech. based miRNA primers, those are readymade, pre-mix (FP+RP), available for most of the miRNAs. Just dissolve and use 1ul for per rxn.
Regards
Saurabh
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I need to stain some fixed/permeabilised cells (platelets) with annexin V conjugated to FITC. Will I be able to pick up fluorescence signal? I am not trying to study annexin V, I just need to stain the platelets to read at FL1 on the flow cytometer. I do not use CaCl2 in my buffers.
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Kanika Arora is correct; Annexin V will not bind without Ca+2 present at 5 mM. Diluting the Ca+2 leads to loss of signal. And there are frequent problems with staining permeablized cells. B-D does make a permeablization/fixation kit that seems to work well.
But I am curious at to why you are using Annexin V to light up your platelets. Annexin V binds to phosphatidyl ethanolamine [PE], which is on the inner cell membrane. It only migrates to the outer cell membrane during apoptosis due to loss of membrane polarity.
Wouldn't it be easier to visualize your platelets in FL1 with anti-CD41 FITC or anti-CD61 FITC?
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A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. If the number drops to around 55, what would you advice to increase it?
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Dear Dr. Ahmed T. Hussein , Thank you for this interesting question.
Let me share our experience.
In India, the people suffering from Dengue, used to take papaya leaf juice (as fresh) or papaya leaf tea ( papaya leaf used instead of tea leaf)- this is in addition to normal medical treatment.
Though the platelet count comes down, it never goes down to the extent of a life threatening situation.
Personally, I used to take once in a while - when I have body pain - it acts as a natural pain killer.
Report:
It has been proved both in modern medicine and Ayurveda that papaya leaf juice is beneficial. 30ml of fresh papaya leaf juice helps in increasing the platelet count and, therefore, in the treatment of dengue.
Thanks and warm regards
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Hello!
I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation.
I will use the ELISA from R&D #DY795.
I have found different buffers to lyse plateles (e.g., TNE,Triton alone, RIPA [with and without SDS] ) but I am still not sure which one would disrupt the membranes of the platelet and the granules.
I have been trying to fine a good reference about the membrane of the granules but I cant find any. So my questions are:
1- Have anyone tried to lyse platelets using different buffers and compare the results?
2- are the membranes of the platelets the same as the granules? (i.e., if a buffer can lyse the cell wall would it have the same effect in granules?)
3-. Have anyone worked with this ELISA or know if it detects denatures PF4 in case I choose a buffer that might denature my protein?
4. Related question... Can PF4 be phosphorylated or cleaved so it justify using protease and/or phosphatase inhibitors?
Thanks in advance for your answers!!
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you might try passing the platelets through decreasing bore size (diameter) needles. Mechanical stimulation is the old man's trick to degranulate platelets. ADP sounds like a great, more physiological stimulator.
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I am trying to visualize alpha and dense granules in platelets. However, it seems like the granules were not stained well. Is there any way I can improve my staining technique to obtain better contrast for better looking images? Below is the staining technique I have used:
1. Uranyl acetate (15 min)
2. Ultra Pure water (3 min)
3. Ultra Pure water (1 min)
4. Lead citrate (7 min)
5. Ultra Pure water (2 min)
6. Ultra Pure water (2 min)
7. Let dry for TEM
Uranyl acetate and Lead Citrate were filtered with 0.22 micron syringe filter prior to use.
Lead citrate was prepared by dissolving 5mg of lead citrate in 1mL of 4.5mM NaOH.
Is it better that I prepare lead citrate using sodium citrate and lead nitrate?
Attached are some of my TEM images from resting platelets. Thank you in advance!
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Thank you Muhammad Irfan I will check my fixation technique!
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My lab is recruiting qualified individuals in the area of thrombosis/platelet biology signaling. No prior experience working with platelets is needed as full training and mentorship will be given. Strong skills in biochemistry, molecular biology, and/or animal models of vascular surgery would be advantageous. The surrounding basic research environment is exceedingly rich with 40 investigators publishing highly impactful studies, and my work at the Cleveland Clinic gives us access to patient data and specimens through institutionally-approved protocols. Our department was ranked #1 for the last 25 years in cardiovascular medicine. Please send me a message if interested.
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I have made considerable studies on neonatal thrombocytopenia in Nigeria and will gladly join your team to investigate more on newborn platelets. My PhD thesis was on aetiology and pattern of neonatal thrombocytopenia at birth in Delta State, Nigeria. Other similar publications include
1. prevalence of thrombocytopenia at birth among apparently healthy newborns in Delta State, Nigeria
2. Alterations in platelet-to-Lymphocyte ratio and some white blood cell indices in neonatal thrombocytopenia
3. Submitted for publication: Some maternal and neonatal risk factors associated with early on-set neonatal thrombocytopenia in Delta State, Nigeria
4. Submitted for review: Variation in some haematological parameters in the newborn at different locations in Delta State, Nigeria
I have over 12 other publications in reputation journals.
My research experience with add value to your Lab
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Question 1: I would like to isolate platelets without activating them beforehand and also at the same time make sure that I can analyse them in non-aggregated forms (i.e. Individual platelets). Will that be possible, and if so are there particular protocols/methods I can refer to?
Question 2: What about achieving the above platelets isolation from blood samples of patients with disease associated with platelets activation and aggregation (e.g. heart disease patients)? Is there a way to ensure that the resultant isolate contains platelets that are not aggregated or adhered to each other or other particulates?
Thank you for taking time to read my question and for providing answers/advice!
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For both questions, the answer is the same. Yes you can. The important thing is to anti-coagulate the blood.
We usually collect whole blood into acid- citrate-dextrose (ACD, 3.2%) sterile tubes. Depending on your read-out you can use this blood for flow cytometry, alternatively you can subsequently isolate the platelets.
Briefly, the whole blood needs to be first centrifuged at 150xg for 20min at room temperature (RT). The platelet-rich plasma (PRP) then needs to be removed and PGE1 was added to the PRP to prevent exogenous platelet activation. The PRP is then centrifuged at 400xg for 20min at RT. The platelet pellet was re-suspended in warmed (37oC) PIPES saline glucose (PSG).
More details can be found here:
PMID: 32573711
PMID: 31366617
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Hi, I am working with TRAP-stimulated and unstimulated platelets in PRP
Right now I am titrating the antibody CD62P but I have several questions about how to analyze my data to select the best dilution.
1.Do I have to select a different dilution for stimulated and unstimulated platelets? I know CD62P mobilize to the platelet surface after activation. So I wonder if the dilution I select for the unstimulated platelets might not be enough to stain platelets after activation.
2. I already have my data for 1:80, 1:160, 1:320, 1:480 and 1:640 dilution for stimulated and unstimulated. I know the geometric mean fluorescence intensity (MFI) for my positive and negative events, the separation and staining index. But, is this enough to select the best dilutions? What should I be comparing? and, can I compare the MFI (either + or -) from the unstimulated to the stimulated?
3- Is it a big difference to use the geometric mean over the median for analyzing the fluorescence intensity?
I don't know how is the best ways to present my data
Thanks for your help.
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Hi Liliana,
1. Make your titration with activated platelets, normally you will not see any positive result for CD62P on unactivated platelets. but because you are using PRP there will be low level of activation due to centrifugation and preparation phase.
2. Your dilutions seemed quite high to me for antibodies to be used on flow, are you using a primary antibody? In this case, our platelets will be even more stimulated while you are preparing for secondary antibody. You can decide through the amount that does not effect the staining index, best is to make a lineer graph to see where you obtain the same results (mean/median channel) with suggested quantity.
3. To use geometric mean or median depends on the cytometry you are measuring, but as long as you are using the same parameter for all your data in this research you will be fine. The best way to present your data would be following MiCyt Guidelines available at below links:
With best wishes,
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We want to use animal platelets instead of FBS.
I want to know which animal blood is the best candidate to have the platelet removed, and by the best candidate, I mean having more growth factors.
I really appreciate any help you can provide.
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Mouse platelets were most sensitive to both agonists. Unlike in man and dog the maximal response to ADP was greater than to thrombin in mouse, rat and guinea pig. P2Y(12) blockade was in all species equally effective as ADP removal in inhibiting thrombin-induced platelet activation whereas P2Y(1) blockade was almost ineffective.
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We study cultivated meat, and there is a question about the relation between platelet number and growth factors and the relation between platelet size and growth factors?
Thank you for your help in advance
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GF biomarkers act on stimulation of PLT for its functions
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Hello,
Please help me know if the platelet size is bigger. Does it mean it has more growth factors or more microtubules within it?
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Daer Asghar!
Please you look at this article:
The microtubule cytoskeleton
PLT shape and integrity are maintained by a well-defined and highly specialized cytoskeleton composed of an intricate system of molecular struts and girders that also regulate PLT size. Likewise, released PLTs contain 10- to 20-fold increased numbers of peripheral microtubule coils, relative to normal controls, that are often organized like balls of yarn rather than rings, which may explain why the majority of these cells have a spherical rather than the discoid form. The inability to further constrict proPLT tips before abscission resulting from bending energies determined by the cortical microtubule band could account for the increase in terminal PLT size.
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Hello,
Which one is more important, the number of platelets or the size when it comes to causing cell proliferation?
Please tell me if the platelet size is larger. Does this mean it has more growth factors or more microtubules?
I really appreciate any help you can provide.
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Mega Platelets are pre mature one
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We are studying cultivated meat, and there is a question about platelet ratio to plasma in bovine.
Thank you for your help in advance
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The benefits
The protocols
Recommendations
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There are several articles on the use of antiplatelet therapy in acute coronary syndrome.
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I have tried to isolate platelet total RNA from the human and sheep blood using TRIzol reagent. After isolation of platelets from the blood samples, total RNA extracted, and quantified using NanoDrop. The RNA concentration was around ~180-230 ng/ul ( 260/280 = ~1.87). The integrity of total RNA was assessed on an agarose gel, but I did not find any RNA bands over the gel except ladder. What could be the possible reason for not appearing RNA bands/smears on the gel? Suggestions and guidance will be highly appreciated.
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Thank you Joyleen Maryann Fernandes.
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Hello dear colleagues! Im looking for a simple method to remove platelets from blood samples. Gradient centrifugation doesnt work well because platelets have same density as mononuclear cells, and i need all fractions of leukocytes. Maybe there is solution that selectively lyse platelets or some other cheap methods?
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Hi,
I am trying to isolate platelets from ovarian cancer ascites. In literature I didn't found protocols for the isolation of platelets from ascites. The only protocols/kits are disponiles for whool blood. Can anyone help me?
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I would like to measure membrane blebbing in mouse platelets. Typically, other cells were shown to undergo blebbing using microscope imaging techniques. But, imaging of mouse platelets has been tricky. I was wondering if anyone knows of any other methods that are available to measure membrane blebbing?
Any help is much appreciated.
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Hi Malcolm,
Thank you very much for the suggestion. I will look into this kit.
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Hello all. I am trying to isolate buffy coat from whole blood (sodium citrate), and planning on cyropreserving it for later analysis (~3-5 years). As to what analysis would include, we are leaving it as open ended as we can, but mostly am thinking of extracting DNA (Qiagen or home extraction) and looking at epigenetics.
What freezing media is typically used for this type of storage? Would a standard 90% PBS and 10% DMSO work, and what are the drawbacks.
Additionally, I read about the potentiality of platelets clumping and potentially interfering with the sample. Does anyone have any advice on purifying platelets from the buffy coat on that end? Thank you all very much.
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firstly, to minimize the platelet contamination from PBMCsw, best way is multiple washing with PBS at low speed for longer time.
And to cryo-preserve buffy coat, you should use 90%FBS+10% DMSO, but if you only going to extract nucleic acid from cells, Using 90% Complete medium (90% RPMI + 10% FBS) with 10% DMSO may also work.
Few people use lower concentration of DMSO as, DMSO hinders in the downstreaming process (DMSO is know inhibitor of PCR). SO, you may use as low as 7% DMSO.
All the best.
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Recently a six part Sysmex XN -350 was installed in our laboratory . On inquiring regarding MU for all parameters, the application specialist mentioned that whole blood was diluted with Cell Pack DCL and the diluted blood was analysed 10 times and the factor was calculated . Limit of quantification . it was calculated as 0.02 for Total leukocyte count (TLC) , 0.01 for Erythrocyte counts (RBC), 0 for Haemoglobin (Hb) , 0.1 for Haematocrit and 2 for Platelet counts.
I am not very convinced as to how it need to be expressed whether as in India we express TLC in terms of X 1000/ microliter ; RBC as X million / microliter and Platelets as 100000/ microliter
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Please go through the article "Basics of Estimating Measurement Uncertainty" by
Graham H White.
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We have observed a decreasing median fluorescence intensity (MFI), and recently extracted data from seven years of analyses (se attached figure) showing a very clear decline in fluorescence intensity. We do not think this has to do with reagents or antibodies. Could it be the red laser declining in activity? Could we somehow compensate (eg. by increasing voltage in the flow cytometry protocol)?
Background: we are running platelets and detect a panel of platelet surface glycoproteins using labelled antibodies. We use a Navios from Beckman-Coulter
Any suggetsions is appreciated.
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This happens with every instrument based on laser, as lasers have their defined life (few thousand hours). So, at voltage "X", you get MFI "999" as the time ( consumption hour) passes, your laser weakens so you give "X-x" voltage, so it leads to decreased MFI.
With time, you need to increase voltages to make a constant incident light, which further will maintain the constant MFI.
There are certain beads (Flosser beads), which maintain the system for you. Once you run the beads and set the voltage, it set it for you.
Anything you find any changes in MFI (suspect of power of incident light), u need to run Flowset beads (according to manufacture protocol) to automatically set voltage to gain similar output.
Hope, it was helpful.
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Hi everyone,
I want to do separation of PBMC's from blood tubes and I want to put them together, in which step of that process it's recommended to pool?
I understand that maybe it will be good to do that after I'm washing the platelets and only then mix the cells pellet.
I can mix 2 blood tubes together from the beginning?
Thank you!
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If the problem is the total volume in the tube, you could mix after the first centrifugation, when you take the "cloud" of PBMCs... All from the same person I suppose :D
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I would like to extract membrane proteins of platelets, but I can't find more detailed protocols. How should I extract without a commercial kit?
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A brief literature search will enable you to find papers describing methods for preparation of platelet membrane proteins. Here are a few examples:
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How do we calculate platelet distribution width in percentage from femto-litter. I have some data in fL and some in %. How to inter convert these two?
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I also have some PDW data in % and wish to convert to fL.
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Hello everyone,
I would like to study the adhesion/aggregation of platelets on different supports, but I was wondering what would be the 'less worse' option for blood collection?
Heparin and EDTA-coated tubes will affect the platelet aggregation properties, but non-coated tubes won't be usable...
Thank you for your help!
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Both can be used. Heparin is batter
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I want to evaluate platelet marker. While running whole blood (10uL), stained antibody on flowcytometer on log scale o Forward scatter and side scatter, find a "L" shape population. I am not able to differentiate platelets and RBC on Forward and side scatter plot (like we do differentiate Lymph, mono, neutro on fsc/ssc plot when we run lysed blood?)
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Gating from the CD41 or CD61-SSC dot plot, which can effectively remove the interference of debris and impurities.
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Ive been using sodium citrate and acd-a collection tubes to spin blood and extract platelets for PRP. The collection tubes I use do not have the separating gel. Ive noticed that in order to get all the platelets sitting right above the RBC I always end up extracting quite a bit of RBC. Im worried that the RBC will hinder the results after injection. Thanks everyone.
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RBC in PRP is a contamination and it should be free of RBC.
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Hi everyone, i have an experiment to be done using platelet membranes. I know that platelets can be isolated from peripheral blood, but then, are there any other ways by which i can still obtain them...like, are they available for purchase?
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Due to the small period of conservation (under 5 days) of the platelets, it's very difficult ( not impossible) to find them outside your place, as the colleagues propose, the best way to get them functional is by contacting the nearest blood bank in your place, however, when you obtain them ensure strict transport procedures like continues agitation in temperature between 21 to 22 C°, in the other side confirm whether you need the platelets functional or not, because as you mentioned above you need to study their membranes, in this case, I think you should do the study on both periods: under 5 days and above 5 days and compare. If the results are the same in this case you can easily get them after 5 days.
Good luck
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Hi all,
Our lab does platelet aggregation studies in humans and pigs both in washed platelets and PRP. After collecting washed platelets we traditionally have normalized the count using an Advia hematological analyzer, however, it is currently undergoing maintenance. Our lab has an Attune NXT flow cutometer, so I was wondering if there was a good method to accomplish the same using that.
Thanks so much in advance for your help!
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Dear you can use other Haemtology autoanalysees to do it.
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Has anyone experience with the FacsVia Flow Cytometer from Becton-Dickinson ? I have had a demonstration few days ago and I'm interested in blood component quality control and in research on platelet concentrates.
It works with leucocount and plasmacount kits on a blood component quality control software. It is also equiped with a "research" software to develop specific analyses.
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HI André Rapaille, this is very good compact equipment with 2 laser 4 fluorescent detectors. we have used this equipment and working on both the software (Research and clinical). Do let me know on parvindsingh1995@gmail.com for any specific query.
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Im planning on using a double centrifugation with ACD-A blood collection tubes. to achieve a high concentration of platelets. I've read that the low ph does not allow the platelets to function correctly. What can I add back to the PRP to raise the PH to 7.4-7.5?
The PRP will be used for injection into soft tissue.
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If you are going to inject them I believe the body will take care of necessary pH adjustments.
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We are trying to coat the substrate of PMMA by using HA in order to collect high concentration of platelet in Platelet-Rich Plasma and preventing the platelet adhesion on the surface of the substrate.
We need to overcome the hydrophobic interaction of the substrate (PMMA) by modifying the surface with the proper solution in which this solution can not affect or contaminate the platelet when we collect them because we are dealing with human blood.
We used NaOH but the HA still not coated well or spread on whole the substrate surface.
So, if there is anyone who dealt with such a thing like this can please help us.
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Hi, try preliminary cold plasma treatment with oxygen or air.
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we are using hanstech oxygraph system in our lab for respirometry study in platelets and isolated mitochondia.
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Thanks alot sir for precious suggestions!
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Hi everybody, I have to isolate MSC from frozen vials of splenic mononucleated cells and splenic CD34-depleted fraction. Do you have any suggestion regarding cell-seeding concentration? shoud I use different concentrations for the two types of frozen sample?
Note that my culture media should consist of alpha-mem added with 10% human platelet lysate.
Many thanks for support!
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many thanks Emmanuel (and sorry for the delay!)
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There is a clot (likely looks like a platelet or fibrin clot) in one of the three bags after thawing of cryopreserved lymphocyte stored for Donor Lymphocyte infusions (Cryopreserved a month ago). The recovery of CD3 cells in the bag is within acceptable limits. There were no clots seen in any other previously thawed bags. The ACD ratio during the procedure and final ACD volume in the product was well maintained.
What could be the reason for this clot ?
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Mononuclear cells sometimes get activated in thaw cycle and start pro coagulant activity. This paper is helpful.
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Transfusion related acute lung injury (TRALI) is a major cause of transfusion related death. Within 6-72 hours of transfusions of whole blood, packed red cells, fresh frozen plasma or platelets TRALI can develop. What could the measure/measures that might help in preventing the development of TRALI?
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Thanks Emmanuel Ifeanyi Obeagu, Dharmesh Chandra Sharma, Gayle Haider for your remarkable comments.
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I want to isolate monocytes from platelet apheresis but I have some doubts about it:
- After platelet apheresis there is a sediment in the bag (WBCs) so how can I select it and isolate the monocytes? I think Ficoll Paque is a good method but that isolates lymphocytes and monocytes at the sime time
- Once isolated the monocytes, how can I differentiate them to macrophages? Is it ok to use M-CSF kits?
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Hi Christian
There is a cheeky way to collect white cells including monocytes from platelet apheresis sample. If you have a leucocyte depletion filter for platelets, put the apheresis sample through it and then flush the white cells out from the filter using saline. You could then do ficol sedimentation to get the mononuclear cells. Good luck.
Siva
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PRP injections accelerates healing of chronic non-healing ulcers by stimulating tissue regeneration through release of Platelet derived growth factor (PDGF), epidermal growth factor (EGF), platelet derived angiogenesis factor and platelet factor 4. There are reports that platelet also exerts antimicrobial effects. How this antimicrobial effects are exerted?
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Part of the answer comes to how the PRP is prepared
Neutrophils phagocytose activated platelets so it is useful to exclude them from your PRP. However they also phagocytose bacteria.
PRP can be applied topically to the ulcer and will aid the healing process. It can also be applied to burns and will help stop secondary infection as well as speed up the healing process. It is one area of use where PRP that is prepared to include neutrophils has apparently been shown to be more effective since the beneficial effects outweigh the platelet phagocytosis effect
I have only heard this as I have not used it on ulcers/burns myself but it certainly makes sense!I cannot give you references
I have also heard that the PRP can be injected into the ulcer as well as applied topically
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We have observed decreasing of platelets count at dengue patients.
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Dengue virus does not attack the blood platelets. The latter are destroyed due to autoimmune reaction induced as a result of infection by dengue virus.
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Hello all-
I am looking for a platelet specific marker to help me identify platelet-leukocyte aggregates in pigs using flow cytometry. I have a good pig specific CD45 antibody that I have validated and works well. For PLTs, however, I cannot find pig specific antibodies and have been trying several directed toward human antigens - specifically CD42a (ALMA.16 clone) and CD42b (HIP1 clone), but neither one has worked. I was able to find a pig specific CD61, but this is not specific to platelets and binds to granulocytes as well. I have seen reports that CD42b (clone AN51) works in pig PRP - and will try this next (although I would prefer something that works in whole blood).
Any thoughts or suggestions?
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First, I would try to make sure you still have platelets in your flow samples. We focus on PMNs and PBMCs and find platelets are so often washed off in sample prep (and aggregates filtered out) we don't even consider them. Concentrate your flow samples (after all filtration, etc.) and have a look with a light microscope to see if you have any platelets left. If your marker is working for platelets but also granulocytes, you should be able to separate those by FSC-SSC.
I'm sure you've seen this, but just in case...
Also, you can always try to find them by a process of exclusion - find a granulocyte specific marker, lymphocyte and monocyte specific marker and platelets should be what you have left.
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Hello, Im going exposure platelets to Aspergillus fumigatus conidia. In this step, I want provide a suspension of platelets then transfer to culture media plates but my problem is that i dont know what kind of plates is necessary for plateles culture ? whether it is essential to polyester plates for adhering them?
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Dear Bahare,
Platelets are "pieces" of megakaryocytes and have no nucleous. So, it is impossible to cultivate them.
Regards,
Nelder Gontijo
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Hi everyone. I would like to ask your opinion about storage conditions of biological material such as blood parts (platelet lysate). Our ultra freezer has dropped its temperature due to power loss from -80 to -60 for about 12 hours. Do you think this could affect stored biological material such as blood parts i.e platelet lysate?
Thank you in advance.
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Thank you very much for your answer.
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Would the following results for CBC WITH DIFFERENTIAL be representative of a pregnant or non pregnant female. If pregnant, why? And approximately what gestation period.
WBC 10.7 K/cumm
RBC 4.01 M/cumm
Hemoglobin 12.8 g/dL
Hematocrit 37.7%
MCV 94.0 fL
MCH 31.9 pg
MCHC 34.0 g/dL
RDW 13.2
Platelets 309 K/cumm
Mean Platelet Volume 9.2 fL
Platelets Distribution Width 9.4 fL
Neutrophil % 69%
Neut. Absolute 7.4 K/cumm
Lymphocyte % 23 %
Lymph. Absolute 2.5 K/cumm
Monocyte %. 6%
Mono. Absolute 0.6 K/cumm
Eosinophil % 2%
Eos. Absolute 0.2 K/cumm
Basophil % 0%
Baso. Absolute 0 K/cumm
additional
BUN/CREATININE/LYTES - Details
BUN 13 mg/dL
Creatinine 0.7 mg/dL
Sodium 139 mEq/L
Potassium 4.3 mEq/L
Chloride 105 mEq/L
Carbon Dioxide 25 mEq/L
Thank you
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interested
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I am running a Dynabead experiment to pull out certain extracellular vesicles from solution. I have 1x10^10 EVs in 100ul, from this I use CD41 (3ug/ml), which in theory should bind to the platelet EVs. (incubation 2 hours). The beads are added (50ul, incubation 30 mins) and should then bind to the CD41, which then cause a decrease in EV number.
However, every time i run this, im not getting a decrease, even though i can confirm CD41 on flow cytometry, has anyone got any tips for the dynabeads or am i missing something.
Thanks for the help
Jamie
(sorry if this sounds confusing, didn't know how to describe the issue)
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Hi Jamie,
Isolation of extracellular vesicles can be a challenge. There are some factors in such a workflow that is important to pay attention to when designing the protocol. One such critical factor is the amount of beads used.
If the downstream application is flow cytometry it is important to use as few beads as possible. typically we use in the range of 40 µL Dynabeads (from 1x10e7 beads/mL vial) in a 100 µL sample volume. Those few beads will capture max number of exosomes/bead = strong signal in flow. However, given the low number of beads used - this is not isolation since most of the exosomes will be remaining in the sample.
For isolation and downstream applications such as western blot the use of beads is completely different. here, as many beads as possible is required in order to get a good WB signal (those beads will give a very low flow signal). Here we typically use 40 µL Dynabeads (from 1.3x10e8 beads/mL vial).
Other factors that is important to pay attention to is the incubation time. Extending the incubation time - even to over night increases the capture significantly.
Feel free to get in touch at ketil.pedersen@thermofisher.com
kind regards
ketil