First, we harvest cells or tissues and prepare a single-cell suspension. We will then transfer the single-cell suspension to a 96-well plate, tube, or polystyrene round-bottom tube, depending on the number and volume of cells used.
Materials Required:
Cell suspension polystyrene round-bottom 12 75 mm2 Falken tubes in a 96-well plate (or any vessel compatible with your centrifuge).
Suspension Wash buffer (PBS, 5-10% fetal cell serum (FCS)).
Optional red blood cell lysis buffer (e.g., R377982).
Step 1
It takes about 20 minutes.
Harvest and wash cells according to the guidelines.
1.1 For blood samples, we recommend incubating the samples with erythrocyte lysis buffer (e.g., R377982).
Tip 1: Red blood cell lysis buffer lyses red blood cells, which may interfere with the analysis of white blood cells (nucleated cells).
Tip 2: Prevent cell damage by avoiding air bubbles, vortexing vigorously, aspirating the entire solution during buffer changes, and over-centrifugation.
Determine the total number of cells and check cell viability.
2.1 In general, the survival rate should be 90-95%.
Centrifuge and resuspend the cell samples in ice-cold suspension buffer.
3.1. Centrifuge at approximately 200 g for 5 min at 4 °C, 32 Recommended suspension cell concentration: 05 1 106 cells ml.
Tip: Rotation time and speed may need to be optimized. In general, cells should be centrifuged well to remove the supernatant, but in order not to cause cell loss, the centrifugation force should not be too strong to prevent the cells from being difficult to resuspend.
Warning: Higher cell concentrations may clog the flow cytometry system and affect resolution.
Continue staining with reactive dyes.
Because dead cells are prone to non-specific binding to antibodies, it is important that these cells are excluded from the analysis. The use of viability dyes allows us to distinguish between live and dead cells and exclude dead cells during data acquisition and analysis.
DNA-binding dyes (e.g., 7-AAD, DAPI, and TOPRO3) are commonly used as reactive dyes for staining dead cells in live cells, as they are unable to penetrate the cell membrane of living cells. The damaged cell membrane in the dead cells allows these dyes to come into contact with the DNA, bind to the DNA, and fluoresce.
However, these dyes cannot be used for live-dead staining of fixed cells, otherwise the cell membrane of all cells will be compromised. In this case, we must use an amine-reactive fixable cell viability dye.
Materials Required:
Reactive dye. Examples of non-fixable dyes: 7-AAD, DRAQ-5, DRAQ-7, or DAPI
Fixable dyes.
Suspension: Wash buffer (e.g., PBS, 5-10% fetal cell serum (FCS)).
Step 1
Cells are stained with a viable dye.
1.Incubate the cells with dye at 4 °C in the dark according to the manufacturer's instructions.
Caution: Keep fluorophores in the dark to avoid photobleaching.
Tip: Choose a dye whose emission spectrum does not overlap with the fluorophore used for immunostaining.
Wash the cells twice with wash buffer.
2.Spin the cells down (200 g, 5 min, 4 °C), remove the supernatant, and resuspend the pellet after each wash.
Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Continued blocking when extracellular targets are detected, or fixation and permeabilization of intracellular targets.
When staining intracellular targets, we must perform additional fixation and permeabilization steps. Structures that retain intracellular proteins need to be fixed. Permeabilization disrupts cell membranes, allowing antibodies to enter the cell and stain intracellular targets.
When staining an extracellular target, we will immediately proceed to the blocking step. When analyzing intracellular and extracellular targets together, we need to perform cell surface staining prior to fixation and permeabilization (see stage 5).
Useful tips for choosing the right method for fixation and permeabilization of intracellular staining:
Antigens close to the plasma membrane and soluble cytoplasmic antigens require mild cell permeabilization without the need for fixation.
Cytoskeleton, viruses, and some enzyme antigens usually produce the best results when fixed with high concentrations of acetone, alcohol, or formaldehyde.
Whether antigens in cytoplasmic organelles and granules require fixation and permeabilization methods depends on the antigen.
Epitopes need to remain accessible.
Materials Required:
Cell suspension. Suspension buffer (PBS, 5-10% FCS).
Fixative (e.g., 1-4% paraformaldehyde, 90% methanol, or acetone).
Permeabilize solution (e.g., Triton X-100, NP-40, or saponin).
Alternatively, you can use flow cytometry fixation and permeabilization kits, which are suitable for most sample types.
Step 1
It takes about 1 hour and 15 minutes.
Fix the cells with the fixative of your choice.
1.1 Centrifuge the cells into a pellet (200 g, 5 min, 4 °C), discard the supernatant, and resuspend the pellet in fixative.
1.2 Incubate the cells with fixative as follows.
Tip: Fixation needs to be optimized for different antigens. Some epitopes are very sensitive to methanol, so if anything goes wrong with the test, try acetone.
Fixer. Time.
1-4% paraformaldehyde (PFA).
15-20 minutes on ice.
90% methanol.
20 °C for 10 min.
100% acetone*
10-15 minutes on ice.
Polystyrene plastic pipes are not suitable for use with acetone.
Wash the cells twice with suspension buffer.
2.1 Centrifuge the cells (200 g, 5 min, 4 °C), discard the supernatant, and resuspend in wash buffer.
Tip: The number of washing steps, spin time, and speed can be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Permeabilize the cells by incubating them with a suitable detergent.
3.1 Centrifuge the cells into a pellet (200 g, 5 min, 4 °C), discard the supernatant, and resuspend in detergent solution.
3.2 Incubate the cells in detergent for 10-15 min at room temperature.
Note: This step is not required if acetone is used as a fixative, as acetone can also permeabilize cells.
Tip 1: The choice of the best detergent depends on the protein and its localization. Powerful detergents such as Triton or NP-40 partially dissolve the nuclear membrane and are therefore suitable for nuclear antigen staining. In contrast, mild detergents, such as Tween 20 or saponins, enable antibodies to pass through the pores without dissolving the plasma membrane, which makes them suitable for antigens and soluble nuclear antigens in the cytoplasm or cytoplasmic surface of the plasma membrane.
Tip 2: The concentration of the detergent should be optimized according to your sample. Tip 3: Permeabilization can affect the light scattering profile of cells on a flow cytometer; Keep this in mind when gating cell populations during detection and data analysis (Phase 6).
Wash the cells twice with suspension buffer.
4.1 Centrifuge the cells (200 g, 5 min, 4 °C), discard the supernatant, and resuspend in the wash slow flush.
Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Perform subsequent blocking steps.
Blocking proteins and Fc domains is essential to prevent non-specific binding of antibodies to cells.
Materials Required:
The choice of material depends on the type of cell being analyzed and, if applicable, the secondary antibody used
FCR blocking buffer (e.g., 2-10% goat serum, human IgG, or mouse anti-CD16 CD32).
Suspension buffer (PBS, 5-10% FCS).
Step 1
It takes about 45 minutes.
Block the FC receptor with blocking buffer.
1.1 Centrifuge the cells into a pellet (200 g, 5 min, 4 °C), discard the supernatant, and resuspend in the blocking slow flush.
1.Incubate the cells with buffer in the dark at 4°C for 30-60 min.
Wash the cells twice with wash buffer.
2.Spin the cells down (200 g, 5 min, 4 °C), remove the supernatant, and resuspend the pellet after each wash.
Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Continue with the antibody incubation.
We are now ready to stain the cells with fluorophore-conjugated antibodies for indirect or direct detection in flow cytometry.
The following procedures can also be repeated and applied to multicolor flow cytometry, where multiple groups of fluorophore-conjugated antibodies are used against different targets. When using multiple groups of antibodies, we should minimize the overlap of fluorophore emission spectra.
Direct antibody labeling
Materials Required:
Conjugated into one antibody. Samples (05-1 cell suspension of 106 ml).
Suspension buffer (PBS, 5-10% FCS).
Step 1
The time is about 40 minutes.
Dilute the bound primary antibody in suspension buffer.
1.1 Recommended dilutions for each antibody are typically provided on the datasheet.
Tip: Titrating your antibody by serial dilutions will help you find the best antibody concentration for your experiment.
Incubate cells in pre-diluted primary antibody.
2.Centrifuge the cells (200 g, 5 min, 4 °C), discard the supernatant, and resuspend the cells in an anti-solution. 2.Incubate at 2 °C at 4 °C in the dark for 20-30 min. Fixed cells can be incubated at room temperature or 4 °C.
Tip: This step may require optimization.
Caution: Keep fluorophores in the dark to avoid photobleaching.
Wash the cells twice with suspension buffer.
3.Centrifuge the cells (200 g, 5 min, 4 °C), remove the supernatant, and resuspend after each wash.
3.2 Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Perform flow cytometry assays as soon as possible.
4.1 If the cells are not analyzed in the flow cytometer immediately after antibody staining (within 1 h) and are not pre-fixed, the stained cells can be fixed at this step (1-4% PFA, 4°C, 20 min). Fixation helps preserve cells for several days, stabilizes light scattering, and inactivates most biohazard agents. Controls need to be pinned using the same program. Note that fixation kills cells and is not compatible with non-fixable cell viability dyes (if these dyes have been used before).
Tip: Get the best results immediately after incubation.
Warning: If you intend to study cells in real time, do not repair them.
4.2 Wash the cells 3 times after fixation and store the cell suspension in suspension buffer.
4.3 Follow the directions for use.
Tip: Keep the cells on dark ice or in a 4 °C freezer until your scheduled analysis time.
Indirect antibody labeling
Indirect labeling requires two incubation steps, first with a primary antibody and then with a compatible secondary antibody. Secondary antibodies, but not primary antibodies, are conjugated with fluorescent dyes (FITC, PE, Cy5, etc.).
Materials Required:
Primary antibodies. Conjugated secondary antibodies.
Samples (05-1 cell suspension of 106 ml).
Suspension Wash buffer (PBS, 5-10% FCS).
Step 1
The time is about 1 hour and 15 minutes.
Dilute the primary and secondary antibodies in suspension buffer.
1.1 Recommended dilutions for each application are typically provided on the antibody datasheet.
Tip: Titrating your antibody by serial dilutions will help you find the best antibody concentration for your experiment.
Incubate cells in pre-diluted primary antibody.
2.1 Centrifuge the cells (200 g, 5 min, 4 °C), discard the supernatant, and resuspend in primary antibody solution.
2.Incubate at 2 °C at 4 °C in the dark for 20-30 min. Fixed cells can be incubated at room temperature or 4 °C.
Tip: Incubation time may need to be optimized.
Wash the cells twice with suspension buffer.
3.Spin down the cells (200 g, 5 min, 4 °C) after each wash, remove the supernatant and resuspend the pellet.
Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Incubate cells in pre-diluted secondary antibody.
4.1 Centrifuge the cells (200 g, 5 min, 4 °C), discard the supernatant, and resuspend the pellet in the secondary antibody solution.
4.2 Incubate in the dark for 20-30 min It is important to keep the fluorophore in the dark to avoid photobleaching.
Wash the cells twice with wash buffer.
5.1 Centrifuge the cells into a pellet (500 g, 5 min, 4 °C), discard the supernatant, and resuspend in the wash buffer.
Tip: The number of wash steps, spin time, and speed may need to be optimized. When using an excess of wash buffer and removing as much liquid as possible after centrifugation, one wash step may be sufficient.
Perform flow cytometry assays as soon as possible.
6.1 If the cells are not analyzed in the flow cytometer immediately after antibody staining (within 1 h) and are not pre-fixed, the stained cells can be fixed at this step (1-4% PFA, 4°C, 20 min). Fixation helps preserve cells for several days, stabilizes light scattering, and inactivates most biohazard agents. Controls need to be pinned using the same program. Note that fixation kills cells and is not compatible with non-fixable cell viability dyes (if previously used).
Tip: Get the best results immediately after hatching.
Warning: If you intend to study cells in real time, do not repair them.
6.2 Wash the cells 3 times after fixation and store the cell suspension in suspension buffer.
6.3 Follow the instructions for data collection.
Tip: Keep the cells on dark ice or in a 4 °C freezer until your scheduled analysis time.
After antibody incubation, we can run our experiments in a flow cytometer. The procedure largely depends on the equipment used, so be sure to check with the manufacturer first.
When surface-stained cells are viable and unfixed or permeabilized, they can be isolated using fluorescence-activated cell sorting (FACS). With FACS, living cells can be grouped into different populations based on their characteristics. We can then perform downstream analysis on the isolated cells.
For more information, please visit our website, which is designed to help you get the best data from your cells.