Cell Capture Imaging Reagent

• What is Immunocytochemistry?
• Immunocytochemistry Procedure: Sample Preparation and Limiting Cell Loss
• Immunocytochemistry Cell Capture Imaging Reagent
• Cell Capture Imaging Reagent Protocol

What is Immunocytochemistry?

Immunocytochemistry (ICC) is a widely used immunostaining technique that offers semi-quantitative means of detecting and analyzing the relative abundance and subcellular localization of target proteins. ICC commonly uses either chromogenic or more sensitive fluorogenic techniques for protein analyses. With the fluorogenic technique, the target protein is visualized using either a fluorochrome-conjugated primary antibody; or by a two-step method where first an unlabeled primary antibody binds specifically to the target protein followed by a fluorochrome-conjugated secondary antibody. ICC differs from immunohistochemistry (IHC) in that intact cells are used without their surrounding extracellular matrix and can be grown in a suitable culture medium. For additional information, please view our immunohistochemistry (IHC) and ICC troubleshooting guide and search our Antibody Explorer for thousands of highly cited antibodies to meet your research needs.

Immunocytochemistry Procedure: Sample Preparation and Limiting Cell Loss

Sample preparation is a critical step in an ICC assay, where results can be negatively affected by cell confluency. Both high cell density and low cell density can affect the experimental outcome, thus achieving optimal cell density (typically 60-80% confluency) is important for obtaining quality data. Furthermore, errors during cell fixation and permeabilization can lead to erroneous or ambiguous data. To further refine ICC data, heat-inactivated serum and a non-ionic detergent or other blocking agents should be added to minimize non-specific binding. When choosing your antibodies, it is important to consider factors such as specificity, host, and application validation, as well as implementing primary antibody, secondary antibody, and label controls.

Immunocytochemisty employs multiple steps and repeated exchange of reagents for cell fixation, permeabilization, blocking, and immunostaining, and buffer washes. This opens the possibility of cell loss at each step, and traditional ICC protocols are not suitable for samples where cell numbers are low. In these precious samples where cell density is low, the impact of routine cell loss can compromise the experiment. To overcome the issue of low cell density in their sample, researchers use different approaches, such as attaching cells on a glass plate with an inert adhesive (e.g., fibronectin, poly-lysine). However, this approach is not well suited for primary cells or cell suspension in culture. Another common approach is to use the Cytospin™ Cytocentrifuge, which can deposit cells evenly onto a glass slide to produce an optically suitable monolayer of cells. However, there are several limitations with the Cytospin™ instrument, including where only one sample can be processed at a time, cells are deposited in a confined field on a slide, and it requires capturing images from multiple microscopy fields.

Immunocytochemistry Cell Capture Imaging Reagent

To improve your cell capture and imaging workflow, we now offer a novel Cell Capture Imaging Reagent (Cat. No. LMR001) that can capture cells and prevent cell loss in modern ICC applications. The Cell Capture Imaging Reagent saves time by eliminating wash steps and is highly effective in applications involving rare cells, low cell count samples, limited access samples, or other scenarios where loss of sample may negatively impact your results. In contrast to other hydrogel-based matrices, the Cell Capture Imaging Reagent allows for alignment of cells to a precision surface for imaging on a standard, unmodified microtiter plate. The Cell Capture Imaging Reagent is optically transparent, permeable to immunoglobulins, and has minimal coloration and autofluorescence. Furthermore, this reagent easily withstands multiple washings, the shearing force of repeated pipetting and enables production of high-quality images regardless of the properties of cell sample or staining reagents. The use of this reagent allows for 100% retention of cells in a small area for rapid high-quality imaging with a single centrifugation step.

Figure 1. Cell Capture Imaging Reagent Procedure

Cell Capture Imaging Reagent Protocol

(Cat. No. LMR001)

1. Wash cells twice with 1x phosphate buffered saline (PBS) (Cat. No. P3813).
2. Re-suspend cells using PBS to achieve a concentration < 1 x 10⁶ cells/mL. (Avoid any protein or serum at this step as it will hinder the hydrogel polymerization process).
3. In a 384- Imaging well-plate (Cat. No. CLS4851), add 40 µL of cell suspension per well. Number of cells can vary depending on the user preference.
4. Add 6.5 µL of Cell Capture Imaging Reagent. Do not mix. Gently dispense using a pipette.

NOTE: This ratio of cell volume to the volume of Cell Capture Imaging Reagent provided above is valid when using 384 Corning High Content microplate with glass bottom plates (Cat. No. CLS4581). If using a different plate with different dimensions this ratio must be re-calculated:

Example:

• 384 Corning High Content microplate with glass bottom plate (Cat. No. CLS4581): 0.0625 cm2
• 96 A/2 Corning High Content microplate (Cat. No. CLS4580): 0.1586 cm2
• Ratio: 0.1586/0.0625= 2.54 Conversion Factor
• Cells: 40 μL x 2.54= 101.5 µL of Cells
• Cell Capture Imaging Reagent: 6.5 µL x 2.54= 16.5 µL of Cell Capture Imaging Reagent

1. Centrifuge well-plate for 5 min. at 500 x g
2. Expose the plate bottom to UV-light for 15 seconds on ChemiDoc with standard UV wavelength, 302 nm

NOTE: Exposure time may need to be adjusted based on UV intensity. UV light must come from the bottom of the well. To optimize UV exposure, a short time course with the correct ratio of PBS to Cell Capture Imaging Reagent can be used. To visualize hydrogel formation, hold 384 well plate up to light to distinguish a slight yellow color and by taking a pipet tip and remove the gel plug from well. Use the shortest possible time frame of UV exposure.

Alternatively, to optimize UV exposure, take any cells resuspended in PBS with the appropriate amount of Cell Capture Imaging Reagent and expose to UV. Start with the shortest time frame followed by steps 7-9 below and by a 10 min DAPI staining (Cat. No. D9542) and washing with PBS. Visualize under a microscope to make sure that the cell retention is high. This same procedure can also be used to determine suitable number of cells to dispense into well.

1. Remove supernatant from each well by gently flicking the plate.
2. Add 1x PBS to each well. Exercise caution not to poke the bottom layer. Discard supernatant by flicking the plate.
3. Repeat step 8 four times.
4. Cells are now ready for immunocytochemistry.

All immunocytochemistry steps can be performed in the well with cells encapsulated in Cell Capture Imaging Reagent. Be sure to keep wells hydrated with PBS or buffer at all times.

Application Note:

Cell Capture Imaging Reagent may also be used with cells prepared for flow cytometry. Once flow cytometric analysis is completed, remaining cells can be encapsulated with Cell Capture Imaging Reagent and visualized by microscopy. For additional information, please view our immunohistochemistry (IHC) and ICC troubleshooting guide and search our Antibody Explorer for thousands of highly cited antibodies to meet your research needs.