Immune Cells

Immune cells include many cells:

Innate immune cells: granulocytes (basophils, eosinophils and neutrophils), mast cells, monocytes (develop into macrophages), neutrophils, dendritic cells (DC is an important antigen presenting cell (APC), which can also develop from monocytes), natural killer cells (NK has the dual characteristics of innate immunity and adaptive immunity and can be retained as memory cells).

Adaptive immune cells: B cells (have two main functions: ① provide antigens to T cells, ② produce antibodies to neutralize infectious microorganisms.) T cells (have multiple functions and are classified by subgroups. T cells are divided into two major categories: CD8+ T cells or CD4+ T cells).

Immune Cell Collection

Immune cell collection usually refers to the collection of peripheral blood mononuclear cells (PBMC). As the name indicates, PBMC refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells (DC) and other small cells.

Lymphocytes include B cells, T cells, NK cells and other cells, which account for the majority of PBMC cells. There are not many articles and operation manuals that separate the two, so here we only give an overview of PBMC cell collection.

PBMC exists in peripheral blood and can be separated from whole blood using cell separation technology. In general, PBMC only accounts for a small part of the whole blood sample (about 1%), and it is difficult to study when it is crowded with other substances. The three most common PBMC separation methods include: density gradient centrifugation, fluorescence activated cell sorting (FACS), and magnetic activated cell sorting (MACS). Each blood separation method has its own advantages and disadvantages.

Density Gradient Centrifugation

Density gradient centrifugation relies on physical properties, such as size and density, to sort cell populations. By placing the sample in a centrifuge spinning at high speed, different cell types are sorted by grouping with particles of similar density. Particles with higher density will fall to the bottom or outside, while particles with lower density will stay in the center or rise to the top. Centrifuging a standard whole blood sample will separate the general layers of blood components, red blood cells concentrated at the bottom of the tube (about 45% of the total volume), a grayish-white coating layer in the middle - containing various white blood cells and platelets (about 1% of the total volume), and plasma - containing the watery fluid that allows blood to flow, along with various proteins and dissolved nutrients and gases (about 55% of the total volume) at the top of the tube.

Density gradient centrifugation also has three methods, depending on the components of the blood collection tube used.

Cell Separation from Blood Collected with Sodium Citrate CPT Tubes

Sodium citrate is an anticoagulant: citrate can bind to calcium ions in the blood to form a soluble complex. Since calcium ions promote coagulation during the coagulation process, a decrease in calcium ion concentration in the blood will prevent blood from clotting.

Steps:

1. Store the plasma collection tubes vertically at room temperature (15℃~30℃) until centrifugation.

2. Gently invert the tubes 8-10 times before centrifugation to remix the cells.

3. Place the centrifuge tubes in a horizontal rotor and centrifuge at 1800 x g (RCF) for 30 minutes at room temperature (15℃~30℃). The speed must be carefully calculated. Do not use the brake during the deceleration process. Care should be taken to ensure that the CPT tubes are correctly installed in the centrifuge.

4. After the centrifuge has come to a complete stop, carefully remove the tubes and place them on a rack.

5. Mononuclear cells and platelets are located in the white layer below the plasma layer (see Figure 1).

6. Without disturbing the cell layer, draw the plasma layer from each CPT tube. If the protocol requires collection of plasma, refer to the protocol specific document.

7. Use a pipette to collect the mononuclear cell layer from each tube and transfer it to a 50mL plastic conical centrifuge tube with a cap.

8. If 3 or fewer (≤3) CPT tubes are collected, place the cell layers collected from all CPT tubes into one 50mL centrifuge tube.

9. If more than 3 CPT tubes are collected (>3), place the cell layers from 2 - 3 CPT tubes into one 50mL centrifuge tube. Do not combine cell layers from more than three CPT tubes into one 50mL centrifuge tube. Repeat this step until the cell layers from all CPT tubes are transferred to 50mL centrifuge tubes, then proceed to the wash step.

Figure 1 CPT tube centrifugation results

Cell Isolation from Blood Collection Using ACD, NaHep, or EDTA Tubes with Prefilled Frit Barriers

Before adding blood, visually inspect the CSTFB to see if there is any fluid on top of the Frit. If there is fluid on top of the Frit, centrifuge the CSTFB at 1000 x g for 30 seconds. If there is any solution remaining on top of the Frit after density gradient centrifugation, remove it by aspiration.

Steps:

1. Dilution of Blood for CSTFB Isolation

The maximum ratio of blood to saline is approximately 2:1. Use one 50-mL tube for every 10 to 20 mL of whole blood (or one 12 to 14 mL tube for every 5 to 10 mL of whole blood). Use as many CSTFBs as required to distribute all the blood for each sample.

(1) Label each CSTFB with the sample identification number.

(2) Use a sterile pipette to add saline solution to each CSTFB: 5 mL saline to a 50-mL CSTFB tube.

(3) Gently mix the whole blood and then transfer the blood to the labeled CSTFB using a sterile pipette.

(4) Using a sterile pipette, rinse each original anticoagulated tube with saline and transfer the rinse solution to the CSTFB, making sure not to exceed the total tube volume (saline + whole blood) limit: the total volume of a 50mL tube should not exceed 30mL.

(5) Carefully cap the CSTFB.

2. CSTFB density centrifugation and PBMC collection

(1) Keeping the tubes upright, gently transfer them to the centrifuge.

(2) Centrifuge at 800 ~ 1000 x g, 15℃ ~ 30℃ for 15 minutes with the brake off. (Some samples may improve PBMC separation by centrifugation at 1000g. If the brake is on during the spin down, it will disrupt the stratification.)

(3) While centrifuging, prepare new sterile centrifuge tubes; this will be the same number of tubes used in the previous step. Label each tube with the sample identification number.

(4) Gently remove the CSTFB from the centrifuge to avoid disturbing the layers.

(5) Centrifuge to separate the contents of the tube (including the Frit layer) into six distinct layers.

(6) Inspect the tube for the following possible problems. Record observations and follow-up actions as required by the laboratory.

①. Plasma + saline solution layer.

②. Visible clots on the Frit after centrifugation.

③. Poor PBMC layer due to centrifugation speed, time, or brake error. The PBMC layer is small and fuzzy, and the plasma + saline layer may be slightly turbid.

④. PBMC layer formed on the Frit due to low red blood cell count or hematocrit.

(7) For each sample, use a new sterile pipette to remove the upper yellowish plasma + saline solution component to a position approximately 1 to 2 cm above the cloudy white PBMC band (located between the plasma + saline solution layer interface and the clear separation medium solution). Discard the plasma + saline solution component according to laboratory policy. (Alternatively, the upper plasma + saline solution fraction can be retained. The cloudy white PBMC band can be carefully pipetted through the upper layer into the PBMC band.)

(8) Use a sterile serological pipette to collect all cells above the PBMC band. Be careful not to aspirate more separation medium solution than necessary.

(9) Transfer the collected cells from one CSTFB to a corresponding, pre-labeled sterile centrifuge tube. Tubes can be pre-filled with saline solution to save time (25 mL saline solution for a 50 mL tube). Follow with the wash steps.

(10) Replace the cap on the CSTFB containing the remaining red blood cells and separation medium. Discard the CSTFB as biohazardous waste in accordance with laboratory policy.

Figure 2 Pre-filled Frit Barrier tube centrifugation results

Manual density gradient medium covering or substrate separation of cells (Ficoll separation)

Ficoll is often used as the main component of the separation solution for separating monocytes. Ficoll is a polymer of sucrose, which is neutral and has an average molecular weight of 400,000. When the density is 1.2g/ml, it does not exceed the normal physiological osmotic pressure and does not pass through biological membranes.

Through Ficoll density gradient centrifugation, the specific gravity of red blood cells and granulocytes is greater than the specific gravity of the separation solution, and they sink to the bottom of the tube after centrifugation; the specific gravity of lymphocytes and monocytes is less than or equal to the separation solution, and they float on the surface of the separation solution after centrifugation, and a small number of cells may also be suspended in the separation solution. By aspirating the cells on the surface of the separation solution, monocytes can be separated from peripheral blood for subsequent culture and testing.

Steps:

1. Blood dilution

(1) Open the cap of the anticoagulant tube.

(2) Label each centrifuge tube with a sample identification number (add 12 to 22mL of blood to a 50mL tube and 4 to 5mL of blood to a 15mL tube).

(3) Transfer the blood to a sterile, labeled 15 or 50 mL centrifuge tube and add enough saline to dilute the blood according to the instructions of the density gradient preparation kit (the maximum ratio of blood to diluent should be 2:1).

2. Density gradient cell separation

(Two methods: (1) overlay, prepare the gradient first and then add the sample; (2) underlay, add the sample first and then the gradient. Close the lid after adding.)

3. Lymphocyte density centrifugation and PBMC collection

(1) Keep the tube upright and gently transfer it to the centrifuge.

(2) Centrifuge at 400 x g for 30 minutes at 15-30°C as outlined in the instructions, with the brake off. (If the brake is applied during the deceleration process, the separation layer will be destroyed. The centrifuge brake must be turned off to ensure clean separation and maximize the recovery of PBMCs.)

(3) Centrifuge to separate the contents of the tube into four layers.

Figure 3 Ficoll density gradient centrifugation results

(4) While the tubes are spinning, prepare new sterile centrifuge tubes. This will be the same number of tubes used in the previous step. Label each tube with the sample identification number.

(5) Remove the tubes from the centrifuge after centrifugation is complete.

(6) If the cell layer is not visible, verify that the centrifuge is functioning properly. Correct any problems you find. Re-centrifuge the tubes. Record the problem and the action taken in the study record.

① If the cell layer is still not visible after re-centrifugation, record, remove and discard the saline supernatant, and continue.

② If the plasma layer is very turbid, it may be difficult to see the interface between the plasma layer and the density gradient medium. Using a 10 mL pipette to remove most of the plasma above the interface may improve lymphocyte collection, e.g., leaving only 0.5 cm of plasma layer. Better positioning of the pipette tip is required for cell collection.

(7) Using a new sterile pipette for each sample, remove the upper yellow plasma + saline solution component to a position approximately 1-2 cm above the turbid white PBMC band (between the pale yellow plasma + saline solution component and the density gradient separation medium solution). Discard the plasma + saline solution fraction as per laboratory policy. (Alternatively, the upper plasma + saline solution fraction can be retained. The cloudy white PBMC band can be carefully pipetted into the PBMC band by carefully inserting a pipette through the upper layer.)

(8) Using a sterile pipette, collect all cells in the PBMC band (the cloudy white portion). Be careful not to aspirate more separation medium solution.

(9) Transfer the collected cells from one conical centrifuge tube to another corresponding, pre-labeled sterile centrifuge tube. The centrifuge tube can be pre-filled with saline to save time. Wash afterwards.

4. Recap the conical centrifuge tube containing the remaining red blood cells/separate. Discard the tube as biohazard waste as per laboratory policy.

Flow Cytometry

An immune cell separation technique that involves the use of a flow cytometer, an instrument that labels different particles based on characteristics such as size, shape, and fluorescent brightness. This method, called fluorescence-activated cell sorting (FACS), allows a sample to flow through a tube and then separates the components into different groups.

FACS requires expensive equipment and properly trained personnel. This technique is useful when sorting different cell samples that require many different populations, but isolating white blood cells using this method is very time-consuming. Typically, samples are first "prepped" or cleaned up before being processed using flow cytometry to isolate specific subsets of the original sample contents, which can reduce the time required for cell sorting because the machine will be processing a smaller, more concentrated sample volume. This can also produce more healthy, viable cells because natural cell death is reduced when processing time is greatly reduced.

Magnetic-activated cell sorting (MACS)

Another method is to bind magnetic beads to target cells and pass the sample through a magnetic field to suspend the cells attached to the magnet. Magnetic-activated cell sorting (MACS) is faster and cheaper than the two methods mentioned above, but it has the highest cell loss of the three methods. Strong magnetic fields can rupture cells, rupture organelles, and mess up samples. This extracellular debris can cause agglomeration and blockage, resulting in lower throughput.

With the continuous advancement of science and technology, I believe that better immune cell separation technologies will continue to emerge, and we will continue to pay attention to related developments in order to provide better services to customers.

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