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Fluorometric Cell-Based ELISA Kit Protocol

Fluorometric Cell-Based (In-Cell) ELISA Protocol | Assay Genie
Assay Genie · Cell-Based ELISA

Fluorometric cell-based ELISA protocol

Measure total and phosphorylated target proteins directly in fixed cells with two-colour fluorescence. This sample protocol covers buffer and antibody-mixture preparation, experimental design, the assay procedure and data normalisation.

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Two-channel fluorescenceTotal & phospho in one wellGAPDH normalisation
96-well
Plate format
2
Fluorescence channels
651/667 & 495/521
Ex/Em (nm)
GAPDH
Normalisation

Overview

Below are the steps for a fluorometric cell-based ELISA kit - buffer preparation, experimental design and the assay protocol. Allow all solutions to reach room temperature before use, and read the whole manual before starting. From the addition of the secondary-antibody mixture onward, keep the plate protected from light.

Buffer & antibody preparation

ReagentPreparation
10x TBSProvided as 10x; washes seeded cells. Prepare 1x TBS by adding 1 volume 10x TBS to 9 volumes ddH2O.
Quenching BufferReady-to-use. Inactivates the endogenous peroxidase activity of the seeded cells.
Blocking BufferReady-to-use. Blocks additional binding sites in each well.
15x Wash BufferProvided as 15x. Prepare 1x Wash Buffer by adding 1 volume 15x Wash Buffer to 14 volumes ddH2O.
Primary Antibody DiluentReady-to-use. Used to dilute the provided antibodies.
Fixing SolutionNot provided. Add formaldehyde to 1x TBS with light mixing. Use 4% formaldehyde for adherent cells and 8% for suspension/loosely attached cells (37% formaldehyde, e.g. Sigma F-8775).
100x Anti-Phospho-Target Primary Antibody100x; dilute 1:100 in Primary Antibody Diluent. Diluted antibody stores at 4°C for up to two weeks.
100x Anti-Target Primary Antibody100x; dilute 1:100 in Primary Antibody Diluent. Stores at 4°C for up to two weeks.
100x Anti-GAPDH Primary AntibodyMouse monoclonal, specific for GAPDH. 100x; dilute 1:100 in Primary Antibody Diluent. Internal positive control for normalisation.
Dye-1 Anti-Rabbit IgG SecondaryDetects target-bound primary rabbit antibodies. Light-sensitive - store and handle in the dark.
Dye-2 Anti-Mouse IgG SecondaryDetects target-bound primary mouse antibodies (e.g. anti-GAPDH). Light-sensitive - store and handle in the dark.
Primary Antibody Mixture P (phospho)Immediately before use, add 50 µL of 100x Anti-Phospho Antibody and 50 µL of 100x Anti-GAPDH Antibody to 4,900 µL Primary Antibody Diluent (one 96-well plate). Mix gently; label "Primary Antibody Mixture P".
Primary Antibody Mixture NP (total)Immediately before use, add 50 µL of 100x Anti-Target Antibody and 50 µL of 100x Anti-GAPDH Antibody to 4,900 µL Primary Antibody Diluent (one plate). Mix gently; label "Primary Antibody Mixture NP".
Secondary Antibody MixtureImmediately before use (one plate), mix 3 mL Dye-1 Anti-Rabbit IgG and 3 mL Dye-2 Anti-Mouse IgG. Mix gently; label "Secondary Antibody Mixture". Light-sensitive - keep in the dark.

Additional materials required

Not provided in the kit but needed to run the assay.

Materials & equipment (not provided)
Fluorescent plate reader with two channels: Ex/Em 651/667 nm and 495/521 nm
Micropipettes covering 1 µL to 1 mL
37% formaldehyde (e.g. Sigma F-8775) or equivalent
Deionised or sterile water
Squirt bottle, manifold dispenser, multichannel reservoir or automated microplate washer
Graph paper or software for logarithmic plots
Absorbent paper or vacuum aspirator
Test/microfuge tubes (>=1 mL)
Orbital shaker
Poly-L-Lysine (e.g. Sigma P4832) for suspension cells

Experimental design

ConsiderationDetail
Cell lineMust express the target protein. Adherent cells work directly; for suspension/loosely attached cells, coat wells with 100 µL of 10 µg/mL Poly-L-Lysine for 30 min at 37°C before seeding and fix with 8% formaldehyde.
Cell number & sensitivityDepends on target expression, cell size, treatment and incubation time. Cells should be ~75-90% confluent.
Cell treatmentTreat with inhibitors, activators or stimulators as required. Include non-stimulated wells on the same plate; keep the plate cover off during treatment. Account for possible cell death from stimulation.
Positive, negative & blank controlsPositive: anti-GAPDH (internal control for normalisation). Negative: Secondary Antibody Mixture only (50 µL Primary Antibody Diluent instead of primary mix). Blank: 50 µL Primary Antibody Diluent instead of any antibody mix. Run all on the same plate.
Accuracy & precisionRun each condition in duplicate or triplicate.

Protocol

The fluorometric cell-based ELISA assay procedure - keep light-protected from the secondary-antibody step onward.

1
Seed 200 µL of the desired cell concentration in culture medium per well of the (sterile, culture-treated) 96-well plate. For suspension/loosely attached cells, first coat wells with 100 µL of 10 µg/mL Poly-L-Lysine for 30 min at 37°C.
2
Incubate cells overnight at 37°C, 5% CO2.
3
Treat the cells as desired. Pipette gently against the well walls to avoid dislodging cells; account for possible cell death.
4
Remove culture medium and rinse twice with 200 µL 1x TBS.
5
Fix cells with 100 µL Fixing Solution for 20 min at room temperature (4% formaldehyde for adherent, 8% for suspension). Seal plates with Parafilm and wear appropriate PPE (the solution is volatile).
6
Remove Fixing Solution and wash 3x with 200 µL 1x Wash Buffer for 3 min each with gentle shaking. The plate can be stored at 4°C for a week. Tap on absorbent paper to remove solution completely.
7
Add 100 µL Quenching Buffer; incubate 20 min at room temperature.
8
Wash 3x with 1x Wash Buffer, 3 min each, with gentle shaking.
9
Dispense 200 µL Blocking Buffer; incubate 1 hour at room temperature.
10
Wash 3x with 200 µL 1x Wash Buffer, 3 min each, with gentle shaking.
11
Add 50 µL 'Primary Antibody Mixture P' to target (phospho) wells and 50 µL 'Primary Antibody Mixture NP' to total-protein wells. Cover with Parafilm and incubate 16 hours (overnight) at 4°C. If target expression is high, 2 hours at room temperature with gentle shaking may suffice.
12
Wash 3x with 200 µL 1x Wash Buffer, 3 min each, with gentle shaking.
13
Add 50 µL 'Secondary Antibody Mixture' to the corresponding wells and incubate 1.5 hours at room temperature with gentle shaking. Keep the plate in the dark for every step from here on.
14
Wash 3x with 200 µL 1x Wash Buffer, 3 min each, with gentle shaking, then rinse once with 200 µL 1x TBS. Keep the plate in the dark during washing.
15
Read the plate at Ex/Em 651/667 nm (Dye 1) and 495/521 nm (Dye 2), shielding it from direct light.

Data normalisation

MethodHow
Target-protein normalisationNormalise the phosphorylated-target RFU using the non-phosphorylated-target RFU: RFU(anti-phospho) / RFU(anti-target). Both RFUs must come from the same read under the same excitation/emission.
GAPDH normalisationNormalise target RFU (phospho and non-phospho) using GAPDH RFU. The ratio anti-GAPDH stimulated / anti-GAPDH non-stimulated estimates relative cell density and can be used as a multiplier when comparing stimulated vs non-stimulated target RFU. Treat these ratios as a qualitative reference for expression levels between wells.

Cell-based ELISA kits & reagents

In-cell ELISA kits and antibodies for total and phospho-protein detection.

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Calculating & Analysing ELISA Data

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FAQs

How is a fluorometric cell-based ELISA different from the colorimetric version?

It uses two dye-conjugated secondary antibodies read on separate fluorescence channels (Ex/Em 651/667 and 495/521 nm), letting you measure the target and the GAPDH loading control - or total and phospho signals - in the same well, rather than a single colorimetric read at 450 nm.

Why prepare Primary Antibody Mixtures P and NP?

Mixture P combines the anti-phospho-target antibody with anti-GAPDH for phospho detection; Mixture NP combines the anti-total-target antibody with anti-GAPDH for total-protein detection. Each is added to its own set of wells.

How do I normalise the data?

Normalise phospho-target RFU to non-phospho-target RFU, and/or normalise target RFU to GAPDH RFU (using the stimulated/non-stimulated GAPDH ratio as a cell-density reference).

Why must the plate be kept in the dark?

The dye-conjugated secondary antibodies are light-sensitive, so from the secondary-antibody step onward the plate should be shielded from light to preserve signal.

Can I use suspension cells?

Yes - coat the wells with 10 µg/mL Poly-L-Lysine for 30 min at 37°C before seeding and fix with 8% formaldehyde instead of 4%.

Running a cell-based ELISA?

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