Exercise 4 - Purification methods for isolating blood cells. Flashcards
Which two methods did you use for isolating immune cells in exercise 4?
1) Lymphoprep
2) Red Blood Cell lysis (RBC Lysis)
Which two tissues did we use for isolating immune cells in exercise 4?
1) Spleen
2) Blood
Why it it important to be able to isolate and analyse leukocytes? And from which tissues would you normally isolate them?
1) Diagnostics
2) In vivo trials
3) Research
Humans: Blood or patient samples (Thymus, Lymph Nodes, Joint Fluid
Mice: Spleen, Lymph Nodes, Organs, Blood
What is RBC lysis buffer?
An ammonium chloride solution that results in osmotic hemolysis of red blood cells. The ammonium chloride is transported across the PM –> Swelling –> osmotic hemolysis
How do we isolate cells using the lymphoprep method?
- Using Ficoll of different densities, you can split blood into different segments. In this case, we used Ficoll with a density of 1,077 g/ml which separates mononuclear cells (monocytes and lymphocytes) from polymorphonuclear cells (granulocytes) and erythrocytes.
- Put Ficoll solution on the bottom, blood on top and spin. Now you get a separation of the two, with the heaviest cells in the Ficoll solution and our lymphocytes on top.
MAYBE: Basophils have a density around 1,075-1,082 g/ml shaped as a bell curve so some will be in the sample, but not many.
How do we isolate cells using the RBC lysis method? And why do we need to do RBC lysis?
- Swelling of RBCs via an ammonium chloride solution results in osmotic hemolysis removing the RBCs.
- Red blood cells (RBC) are a contaminant in flow
cytometry assays that must be removed or lysed in order to properly gate leukocytes. - They can clog up the machine.
What is an advantage of the lymphoprep over the RBC?
If you are interested in staining granulocytes, you need only lyse the RBC.
If you are interested in purifying peripheral blood mononuclear cells (lymphocytes, and macrophages/monocytes only), you must purify your PBMC using a density gradient procedure
What is a CFSE stain and what can we use it for?
- A tool for following cell proliferation. This fluorescent marker is passed down through cell divisions and diluted each time. This way, you can follow cell division generations as peaks on a fluorescent measurement graph.
What did we do in exercise 4?
- Purified leukocytes via two methods, RBC or lymphoprep, stained with Ab for CD3, CD4, CD8 and CD19 and analysed with flow cytometry.
Hvad er CD3?
En del af TCR:CD3 komplekset - med til aktivering af T-celler.
Hvad viser hhv. CD3, CD4, CD8 og CD19?
1) CD3 bruges til at genkende T celler da den er i kompleks med TCR
2) CD4 + CD8 bruges til at differentiere mellem CD4 hjælper celler og CD8+ cytotoksiske T celler
3) CD19 bruges til at genkende B-celler
How can flow cytometry be used for disease diagnostics and treatment?
1) We are able to determine if the sample is missing certain essential cells or has too many by their surface markers e.g., too many T cells can be an indicator of acute lymphocytic leukaemia among other diseases. We could also see if there is an overweight of cells with a certain surface marker, which could inhibit a tumor for example (NKG2A).
2) For treatment purposes, you could sort cells into specific populations using FACS. Later, you could treat these different cells, e.g. sorting by NK cells and making them express more NKG2D receptors on their surface, potentially fighting cancer more readily. Or concentrate missing cells and inject them back into the body.
How can we discriminate between different cell populations by flow cytometry?
- Staining for, and using surface markers specific for that cell type.
Describe any differences between the cell types purified by Lymphoprep and RBC lysis.
1) Lymphoprep yielded more viable cells since it was more gentle than RBC. Much more dead cells and debris in RBC.
2) Lymphoprep seems to have more “unwanted” cells as we see big and granular cells in some of our lymphoprep flow cytometry results.
Describe any differences between cell types purified from spleen and blood and general observations.
Blood: We saw much more T-cells - circulate more readily than B-cells.
Spleen: We saw much more B-cells - makes sense, B-cells go to secondary lymphoid tissues after being formed in the BM. Activation through APC in lymphoid tissues.
General: We saw more CD4+ cells than CD8+ positive cells in both blood and spleen according to our CD4/CD8 stain. Makes sense, as CD8 cells are activated during infection, and CD4 cells help regulate and activate cells. –> CD8 expand during infection and CD4 would be more constant.
