Blood Sample Preparation E-Book
Download this practical guide on the basic techniques of blood sample processing and cell isolation from human blood samples.
Blood is an important source of immune and non-immune cells that can either be used directly in biological assays or further processed to isolate specific cell subsets. The careful choice of protocols and technologies can simplify blood processing, resulting in increased lab efficiency and reduced risk of operator error. Incorporating effective blood sample preparation methods into your workflows can thus help ensure accurate results in your downstream assays.
In this guide you will find information on:
- Sources of human blood samples
- Processing blood samples for downstream cell isolation
- Techniques and principles for isolating cells from blood
- Cryopreserving isolated cells
- Protocols and technical tips
Continue reading for a preview from the e-book.
Page 9, Peripheral Blood Mononuclear Cell (PBMC) Preparation From Whole Blood
PBMC Isolation Using Immunomagnetic Cell Separation
Compared to time-consuming density gradient centrifugation, immunomagnetic cell isolation can be used to isolate PBMCs directly from blood in a single step that can also be automated. For example, the EasySep™ Direct Human PBMC Isolation Kit can be used to obtain PBMCs in as little as 20 minutes by targeting RBCs, platelets, and unwanted cells for immunomagnetic depletion (Figure 4). Untouched PBMCs are simply poured into a new tube and are immediately available for downstream applications such as flow cytometry or cell culture. For additional information on immunomagnetic cell separation, see page 11.
How does EasySep™ Direct PBMC isolation compare to density gradient centrifugation?
- Reduces platelet contamination
Density gradient centrifugation does not remove platelets, which can easily become activated and affect downstream applications. Removing platelets after the initial density gradient centrifugation requires additional time-consuming washing steps. The EasySep™ Direct Human PBMC Isolation Kit conveniently removes platelets during the cell isolation step, so no additional washes are required.
- Reduces granulocyte contamination
Granulocytes in blood samples degranulate and change density over time. This presents a challenge when using density gradient centrifugation to process older blood samples (> 48 hours after collection), as granulocyte contamination of the PBMC fraction may occur. By specifically targeting unwanted cells for removal with antibody complexes and magnetic particles, the EasySep™ Direct Human PBMC Isolation Kit results in less granulocyte contamination than density gradient centrifugation (see Figure 5).
- Enables automated PBMC isolation
PBMC isolation with EasySep™ Direct can be automated using the RoboSep™-S instrument to minimize sample handling and free up valuable hands-on time. By automating all sample labeling and magnetic separation steps, you can perform simultaneous cell isolations from up to four samples to increase sample throughput.
Figure 4. Typical EasySep™ Direct Human PBMC Isolation Profile
(A) Starting with human whole blood from normal healthy donors, the typical mononuclear cell content of the non-lysed final isolated fraction is 98.3 ± 2.8% (gated on CD45). In the above example, the mononuclear cell contents of the whole blood starting sample (lysed by ammonium chloride) and non-lysed final isolated fraction are 27.0% and 98.6% (not gated on CD45), respectively. (B) Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. Representative FSC vs. SSC flow cytometry plots (not gated on CD45).
Figure 5. EasySep™ Direct Human PBMC Isolation Kit Results in Fewer Contaminating Cells Than Density Gradient Centrifugation
PBMCs were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. Cells were counted and analyzed by flow cytometry. (A) Density gradient centrifugation and EasySep™ Direct Human PBMC Isolation Kit resulted in equivalent total numbers of nucleated cells recovered from 24-hour blood samples (mean ± SD; n = 14). (B) Using EasySep™ Direct Human PBMC Isolation Kit to obtain PBMCs from 24-hour-old blood samples resulted in fewer residual platelets (CD41+), red blood cells (Glycophorin A+/CD45- ), and granulocytes (CD66b+) than density gradient centrifugation (mean ± SD; n = 15). (C) PBMC isolation from 24-, 48-, 72-, and 96-hour-old blood samples using EasySep™ Direct Human PBMC Isolation Kit resulted in fewer residual granulocytes than cell isolation using density gradient centrifugation (mean ± SD; n = 3).
Page 10, Polymorphonuclear Cell (PMNC) Preparation From Whole Blood
Figure 6. Protocol for EasySep™ Direct Human PBMC Isolation Kit
Polymorphonuclear Cell (PMNC) Preparation from Whole Blood
Polymorphonuclear cells (PMNCs), also known as granulocytes, are a collection of immune cell subsets with enzyme-containing granules that can be released upon cell activation. PMNCs include neutrophils, eosinophils, basophils, and mast cells. To obtain a specific population of PMNCs from whole blood, you can do any of the following:
- Perform density gradient centrifugation followed by ammonium chloride lysis of red blood cells (RBCs) in the granulocyte fraction. See the protocol on page 22. You can then isolate specific granulocyte populations (i.e. neutrophils, basophils, or eosinophils) using immunomagnetic cell separation technologies.
- Directly isolate granulocytes from whole blood, without lysis or density centrifugation, using immunomagnetic cell separation techniques. For example, EasySep™ Direct kits enable you to quickly and easily obtain pan-granulocytes or a specific granulocyte subset (i.e. neutrophils, basophils, or eosinophils). EasySep™ Direct uses immunomagnetic cell isolation technology for separating PMNCs from whole blood (see page 9 for a comparison between density gradient centrifugation and EasySep™ Direct).
Pages 14-15, Isolation Of Immune Cell Subsets From Whole Blood
Isolation of Immune Cell Subsets from Whole Blood
It is often necessary to isolate specific immune cell subsets from blood for various applications, including research, assay development, and diagnostics. Depending on your needs and available equipment, you can either isolate specific immune cell subsets from whole blood or obtain peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMNCs) first, followed by immune cell subset isolation. There are various approaches you can use to obtain specific cell subsets. The following methods will be covered in this section:
- Obtaining cell subsets directly from whole blood using immunodensity cell separation
- Obtaining cell subsets directly from whole blood using immunomagnetic cell separation
- Isolating PBMCs or PMNCs first (see pages 7 and 10 for more information), followed by immunomagnetic cell separation to obtain specific cell subsets
- Alternative methods for specific immune cell isolation from PBMCs and PMNCs
Immunodensity Cell Separation Directly from Whole Blood
Immunodensity cell separation, also referred to as erythrocyte rosetting, is a negative selection method that uses a combination of antibody-based labeling and density gradient centrifugation. In this method, an antibody cocktail is added to a whole blood sample in which unwanted cells are labeled and cross-linked to RBCs. This results in the formation of complexes called immunorosettes, which are much denser than the mononuclear cells being isolated. During centrifugation, the unwanted cells pellet with the RBCs, leaving the desired cells in a layer above the density gradient medium.
Immunodensity cell separation does not require any specialized equipment beyond a centrifuge, can be easily incorporated into established density gradient centrifugation protocols, and can be used to isolate specific cell subsets directly from whole blood. However, the technique is limited to negative selection, relies on the operator’s blood sample layering technique, and requires a high concentration of RBCs in the starting sample.
RosetteSep™ is a fast and easy immunodensity tool for the isolation of untouched cells directly from whole blood. This approach significantly reduces sample handling time and maximizes convenience by incorporating cell subset isolation into the centrifugation process (Figure 10). RosetteSep™ can be combined with SepMate™ PBMC Isolation Tubes for even faster and easier immunodensity cell separation.
See page 25 for a protocol for isolating specific cell subsets by immunodensity cell separation.
Figure 10. Typical RosetteSep™ Cell Separation Protocol
Immunomagnetic Cell Separation Directly from Whole Blood
Immunomagnetic cell separation is a technique that uses antibody-linked magnetic particles to isolate desired cells from heterogeneous mixtures. Because of its speed and simplicity, immunomagnetic cell separation is one of the methods most commonly used to isolate highly purified populations of specific cell subsets. For more information on immunomagnetic cell separation, see page 11.
EasySep™ Direct is an immunomagnetic cell isolation method that can be used to isolate specific cell subsets directly from whole blood. As described earlier for PBMC and PMNC isolation from whole blood (see pages 7 and 10), EasySep™ Direct may be used to immunomagnetically deplete RBCs and other unwanted cells in a single step without density gradient centrifugation, RBC lysis, or other pre-processing steps that can alter cellular function or increase cell isolation time. EasySep™ Direct kits are available for isolating a variety of specific cell types. The isolated cells are untouched and highly purified, making them ideal for downstream applications like gene expression analysis, functional assays, or flow cytometry.
When using EasySep™ Direct, individual samples of 0.5 - 30 mL can be processed in as little as 20 minutes. To isolate cells from up to 16 samples simultaneously, combine EasySep™ Direct with the EasyEights™ EasySep™ Magnet. For more information on how EasySep™ Direct works, see page 9.
Isolate Cells Directly from Whole Blood
Quickly obtain highly purified cells directly from whole blood by immunomagnetically depleting RBCs and unwanted cells in a single step without density gradient centrifugation, sedimentation, or RBC lysis. Untouched cells isolated with EasySep™ Direct are ideal for downstream applications such as gene expression analysis, functional assays, and flow cytometry.
Immunomagnetic Cell Isolation from PBMC or PMNC Fractions
After obtaining PBMCs or PMNCs from blood products (see page 7 for how to isolate PBMCs and page 10 for how to isolate PMNCs), immunomagnetic cell isolation can be performed on either fraction to isolate target cells from the heterogeneous PBMC or PMNC populations. For example, EasySep™ is a versatile immunomagnetic cell separation technology that enables positive selection, negative selection, or cell depletion to obtain immune cell subsets in as little as 8 minutes. Cell subsets isolated with EasySep™ are highly purified, viable, functional, and ready for a variety of downstream assays. For more information on immunomagnetic cell separation, see page 11.
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