Cell Sample Preparation

Below are some general guidelines to prepare cell samples for use in cell-based assays. Included are examples for microplate (or microtiter)-based and flow cytometry-based applications. Cell preparation protocols should always be evaluated on a per experiment basis and may need to be optimized for individual cell lines.

Mammalian cells: Microplate reader assays (96 or 384 well)

Each cell line should be evaluated on an individual basis to determine the optimal cell density. Poly-D lysine plate can be considered to facilitate cell attachment for non-adherent cells.

Adherent cells

Plate cells overnight in growth medium. Table 1 provides a rough guideline of cell densities per well. In general, assays which require long incubation times (2-3 days) should be plated at a lower initial cell density.

Table 1. Guideline for cell densities per microplate well.

	Examples Assays	96 wells	384 wells
Standard incubation	Calcium, NAD/NADH, membrane potential	40,000 to 80,000 cells/well/100 µL	10,000 to 20,000 cells/well/25 µL
Long incubation	Proliferation, tracking	5,000 to 10,000 cells/well/100 µL	2500 to 5000 cells/well/50 µL

Non-adherent cells

Centrifuge the cells and carefully discard the supernatant (i.e., the culture medium).
Re-suspend the cell pellet in cell growth medium or HHBS. Table 1 provides guidelines on cell densities for resuspension. In general, assays which require long incubation times (2-3 days) should be resuspended at a lower initial cell density.
Transfer resuspended cells into the assay microplate.
Centrifuge the microplate at 800 rpm for 2 minutes with brake off prior to experiments.

Table 1. Guideline for cell densities per microplate well.

	Examples Assays	96 wells	384 wells
Standard incubation	Calcium, NAD/NADH, membrane potential	125,000 to 250,000 cells/well/100 µL	30,000 to 60,000 cells/well/25 µL
Long incubation	Proliferation, tracking	10,000 to 20,000 cells/well/100 µL	5,000 to 10,000 cells/well/50 µL

Mammalian cells: Flow cytometry assays

Each cell line should be evaluated on the individual basis to determine the optimal cell density. For detaching adherent cells from the plate, 0.5 mM EDTA is recommended. Enzymatic reagents (e.g. trypsin, Accutase™) can be considered but need to be tested to make sure the receptor of interest on the cell surface is not affected.

Adherent cells

Plate cells at 400,000 to 800,000 cells/mL in cell growth medium the day prior to use.

Non-aherent cells

Centrifuge the cells and carefully discard the supernatant (i.e., the culture medium).
Re-suspend the cell pellet in 500 µL - 1 mL cell growth medium or HHBS at 500,000 to 1,000,000 cells/mL.

Other cell types: Preparation and Lysing

Below are some general guidelines for preparing/lysing plant, bacterial, mammalian or tissue cell samples. Note that each cell type should be evaluated on an individual basis to determine the optimal cell densities and conditions.

Plant cells

Homogenize leaves with lysis buffer at 200 mg/mL.
Centrifuge at 2500 rpm for 5-10 minutes.
Use the supernatant for tests.

Bacterial cells

Collect bacterial cells by centrifugation (10,000 g, 0 °C, 15 min).
Add 1 mL of lysis buffer per 100 to 10 million cells and incubate the treated solution at room temperature for 15 minutes.
Centrifuge at 2500 rpm for 5 minutes.
Use the supernatant for tests.

Mammalian cells

Remove medium from plate wells and add about 100 µL lysis buffer per 1 to 5 million cells (or 50-100 µL/well in a 96-well cell culture plate).
Incubate the treated solution at room temperature for 15 minutes.
Use the cell lysate directly or centrifuge at 1500 rpm for 5 minutes and then use the supernatant for tests.

Tissue

Weigh ~20 mg tissue and wash with cold PBS.
Homogenize with 400 µL of lysis buffer in a micro-centrifuge tube.
Centrifuge at 2500 rpm for 5-10 minutes.
Use the supernatant for tests.