The document outlines the protocol for performing indirect flow cytometry (FACS), detailing the steps for preparing cell samples, staining with primary and secondary antibodies, and analyzing the samples using a flow cytometer. Key points include the need for specific incubation times and proper washing of cells to ensure accurate results. The document emphasizes optimization and provides links for further resources and the antibody validation project.

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Protocol Series:
Indirect Flow Cytometry
Information on the general steps required for carrying out Indirect Flow Cytometry
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2. Indirect flow cytometry, also known as fluorescence-activated cell sorting (FACS) is a specialised type of flow
cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers,
one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful
scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells
as well as physical separation of cells of particular interest.
In contrast to direct flow cytometry, FACS requires two incubation steps. Firstly with the appropriate primary antibody,
then with a fluorochrome-labelled secondary antibody.
The following slides provide information on the general steps involved. Optimisation may be required.
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3. Preparation of the Sample from Cell Culture
1. Harvest and wash the cells.
2. Determine the total cell number. It is also advisable to check cell viability, which should be no less than
90%.
3. Transfer the suspension to a container suitable for centrifuging.
4. Centrifuge then remove the supernatant.
5. Prepare the cell suspension to a concentration of 1-5 x 106 cells/ml using ice cold PBS, 10% FCS and 1%
sodium azide.
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4. Primary Antibody Staining
1. Add 100μl cell suspension to each tube.
2. Add the quantity of primary antibody recommended by the manufacturer. If necessary, dilute the primary
antibody in 3% BSA or PBS to the dilution recommended by the manufacturer.
3. Incubate at either room temperature or 4°C for at least 30 minutes.
4. Wash the cells 3 times by centrifugation at 400 g for 5 minutes.
5. Re-suspend the cells in ice cold PBS.
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5. Secondary Antibody Staining
1. Dilute the secondary antibody in 3% BSA or PBS to the dilution recommended by the manufacturer.
2. Resuspend the cells in this solution.
3. Incubate in the dark at either room temperature or 4°C for at least 30 minutes.
4. Wash the cells 3 times by centrifugation at 400 g for 5 minutes.
5. Resuspend the cells in ice cold PBS, 3% BSA and 1% sodium azide.
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6. Analysis
1. As soon as possible, analyse the cells using a flow cytometer. If you need to wait longer than an hour, then
fix the cells using either paraformaldehyde (PFA), acetone or methanol (see next slide).
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7. Fixation
1. Apply 100μl 0.01-1% PFA to each sample for 10-15 minutes.
OR
Add 1ml ice cold acetone or methanol to each sample and mix gently. Store at -20°C for
5-10 minutes. Centrifuge and wash twice in PBS 1% BSA.
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8. Download this protocol in a PDF format:
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9. Get Involved with our Antibody Validation Project
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10. Antibody Validation Project
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Receive rewards when you validate our products.
Click here to find out more.