Antibody Specificity and Non-Specific Binding Quiz

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What is the potential issue with using an isotype control antibody in an experiment?

The nonspecific binding can lead to false-positive results.

How can you prevent unspecific binding of the antibody Fc-region to Fc-receptor?

You can block the FC-receptors with a blocking agent like bovine serum albumin or milk.

Why is Alexa-fluor 633 chosen as the optimal election for envoplakin staining?

Because it is a better option than Alexa-fluor 555 for visualization of envoplakin.

What is the potential consequence of nonspecific binding in immunostaining and fluorescence microscopy?

False-positive results. Signup and view all the answers

What can the available microscope contain to ensure proper visualization of the indicated dyes?

The light sources and filter combinations needed for proper visualization of the indicated dyes. Signup and view all the answers

What is the emission peak of DAPI dye?

Approximately 461 nm Signup and view all the answers

What is the emission peak of Propidium Iodide (PI)?

Approximately 636 nm Signup and view all the answers

What is the emission peak of the secondary antibody used for actin visualization?

Around 500 nm Signup and view all the answers

What is the emission peak of the secondary antibody used for envoplakin visualization?

Around 660 nm Signup and view all the answers

Why is it essential to choose a counterstain with minimal spectral overlap with the fluorophores?

To avoid signal bleed-through and maintain imaging specificity Signup and view all the answers

Why is DAPI considered a suitable choice for counterstaining the cell nuclei?

It emits in the blue spectrum, far from the emission peaks of Alexa Fluor 488 and Alexa Fluor 633 Signup and view all the answers

What does spectral overlap with fluorophores cause?

Signal bleed-through and interference Signup and view all the answers

How does DAPI's emission peak minimize interference with other fluorophores?

It is less likely to cause spectral overlap with the emission signals of other fluorophores Signup and view all the answers

What is the purpose of using a counterstain in this context?

To allow clear visualization of the nuclei without interference from actin and envoplakin signals Signup and view all the answers

How does DAPI's emission peak align with the emission peaks of the secondary antibodies used?

It is far from the emission peaks of both Alexa Fluor 488 and Alexa Fluor 633 Signup and view all the answers

What is the purpose of creating gates on negative and positive populations in flow cytometric analysis?

To distinguish between cells expressing the surface marker of interest (positive) and those not expressing it (negative). Signup and view all the answers

How is the stain index calculated in flow cytometric analysis?

The stain index is calculated using the formula: (MFI of positive population - MFI of negative population) / (2 * SD of the negative population). Signup and view all the answers

What is the purpose of creating a forward scatter/side scatter gate in flow cytometric analysis?

To select the cells of interest based on their size and granularity. Signup and view all the answers

What is the significance of determining the optimal concentration of the CD25 antibody in flow cytometric analysis?

The optimal concentration ensures accurate and specific staining of the target lymphocytes without nonspecific binding. Signup and view all the answers

What is the purpose of titrating the CD25 antibody at different concentrations?

To identify the concentration that provides the strongest signal with minimal nonspecific binding. Signup and view all the answers

What is the function of the CD25 antibody in flow cytometric analysis?

The CD25 antibody is used to specifically stain and identify lymphocytes in the blood sample. Signup and view all the answers

What is the purpose of creating fluorescence histograms gated on forward/side scatter in flow cytometric analysis?

To visualize the distribution of cells based on their size and granularity characteristics. Signup and view all the answers

What surface markers need to be detected on adipose-derived stem cells (ASCs)?

CD73, CD90, CD105, and CD31 Signup and view all the answers

What is the expression of CD73, CD90, CD105, and CD31 on ASCs?

CD73, CD90, and CD105 are highly expressed, while CD31 is negative Signup and view all the answers

What is the setting of the flow cytometer used in the lab?

Not provided in the given text Signup and view all the answers

Which antibody and detector should be selected for each surface antigen? Provide an explanation.

CD73, CD90, and CD105 require a fluorescent antibody and a corresponding detector, while CD31 does not require any antibody and detector due to its negative expression. Signup and view all the answers

What is the spillover source to be analyzed?

The spillover source is <a href="https://app.fluorofinder.com/">https://app.fluorofinder.com/</a> Signup and view all the answers

What is the fixable viability dye used in the 'Viability' folder?

FVS570 Signup and view all the answers

How many samples are there in the 'Viability' folder?

There are 7 samples in the 'Viability' folder Signup and view all the answers

What is the surface marker stained in the 'dilution' folder?

CD25 Signup and view all the answers

Where can the information about the optimal concentration to stain the lymphocytes be found?

The information about the optimal concentration can be found at <a href="https://www.bdbiosciences.com/content/bdb/paths/generatetds-document.dk.562442.pdf">https://www.bdbiosciences.com/content/bdb/paths/generatetds-document.dk.562442.pdf</a> Signup and view all the answers