Mike Barnkob:Protocols/Immunology/Flow/Generic protocol

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Generic protocol for flow

Basic protocol for doing flow.

Reagents

• Antibodies
• CD16/CD32 antibody for Fc-block.
• Live-dead stain.
• FACS buffer

Protocol

General notes:

• Create single-stain controls for compensation and remember to leave one control as unstained.
• I do most stains in 96 u-well plates, which makes it faster for washing steps. Before running, samples are transferred to tubes.
• You can do the staining in lower volumes then indicated here, just as long as you don't have too many cells.

Protocol:

• Wash cells and resuspend in 100 μL FACS buffer, with 1:100 Fc-block. Let sit at 4°C for 10 min.
• Wash with PBS, spin at 1500 RPM for 3 min. Flick out supernatant. Resuspend in 100 μL PBS. Add n+1 x 100 μL to single-stain control sample, and pipet out into individual wells. Add 1 μL live-dead stain to all samples and to one control well. Add 100 μL PBS to all other control sampels. Keep in dark at room-temperature for 15 min.
  Note: Most live-dead stains requires that there are no protein be in the media, which is why I use PBS for this step.
  Note: I try and keep time in PBS to a minimum though.
• Wash cells with 100 μL FACS buffer, spin at 1500 RPM for 3 min. Flick out supernatant. Resuspend in either 100 μL with tetramer at 1:10 or antibody cocktail at relevant dilutions. Add individual antibodies to controls. Let sit at 4°C for 10-20 min.
  Note:I do tetramer staining individually from other staining, with a wash step in-between.
• Wash cells with 100 μL FACS buffer, spin at 1500 RPM for 3 min. Flick out supernatant. Repeat once.
• Resuspend in 200-400 μL FACS buffer.
• Transfer samples to flow tubes and off to the machines!