This document outlines the protocol for conducting direct flow cytometry, a technique used for cell analysis, which includes steps for sample preparation, antibody staining, analysis, and fixation. It emphasizes the importance of using fluorescent-labelled antibodies and provides detailed instructions for optimizing the procedure. The document also mentions the availability of the protocol in PDF format and links for further resources.

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Protocol Series:
Direct Flow Cytometry
Information on the general steps required for carrying out Direct Flow Cytometry
St John's Laboratory Ltd St John's Laboratory Ltd @StJohnsLabs St John's Laboratory Ltd
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2. Flow cytometry is a method used for cell counting, cell sorting, biomarker detection and protein engineering. It uses
lasers to enable simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands
of particles per second. Cells must be suspended in a stream of fluid and incubated with fluorescent-labelled antibodies
which detect the expression of cell surface and intracellular molecules. The suspension is then passed by an electronic
detection apparatus. The protocol for this technique is similar to but differs from that used for indirect flow cytometry,
aka fluorescent-activated cell sorting (FACS).
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 using ice cold PBS, 10% FCS and 1% sodium azide. Prepare the cell suspension
to a concentration of 1-5 x 106 cells/ml.
2. Transfer the suspension to a container suitable for centrifuging.
3. Centrifuge then remove the supernatant.
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4. Antibody Staining
1. 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.
2. Incubate at either room temperature or 4°C for at least 30 minutes.
3. Wash the cells 3 times by centrifugation at 400 g for 5 minutes.
4. Re-suspend the cells in 500-1000μl of ice cold PBS, 10% FCS and 1% sodium azide.
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5. 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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6. 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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7. Download this protocol in PDF format:
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8. Antibody Validation Project
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