Jul 22, 2020

Public workspaceFixing cell pellets V.1

DOI
dx.doi.org/10.17504/protocols.io.bg2fjybn
  • 1Wellcome Sanger Institute
  • Cellular Generation and Phenotyping
Verity Goodwin
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DOI: dx.doi.org/10.17504/protocols.io.bg2fjybn
Protocol Citation: Verity Goodwin, Emily Souster, Mathew Garnett, Fiona Behan, Charlotte Beaver, Rizwan Ansari, Adam Jackson 2020. Fixing cell pellets. protocols.io https://dx.doi.org/10.17504/protocols.io.bg2fjybn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2020
Last Modified: July 22, 2020
Protocol Integer ID: 37671
Abstract
This protocol is designed to outline the process of fixing cell pellets in 1.5ml tubes. It has been developed within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute.

Process diagram:



Materials
MATERIALS
ReagentDPBSInvitrogen - Thermo FisherCatalog #14190
ReagentFalcon Round bottomed 5ml tube with cell strainer lidVWR international LtdCatalog #734-0001
Reagent37% FormaldehydeAppliChemCatalog #A0823
Equipment

  • Chemical Fume Hood
  • Microfuge
  • Rainin Single Channel Electronic Pipette LTS E4 1000XLS+
  • Rainin 1200ul tips
  • Pipetboy
  • Stripettes
Safety warnings
Please refer to the manufacturer's documentation and material safety data sheets (MSDS) for the products you are using when following this protocol.
Safety information
Formaldehyde: Toxic if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage. May cause an allergic skin reaction. May cause respiratory irritation. Suspected of causes genetic defects. May cause cancer. Causes damage to organs.

Before start
This protocol must be carried out in a chemical fume hood.
Fixing
Fixing
In a fume hood, prepare a solution of 3.7% formaldehyde in PBS
Safety information
Chemical safety: Formaldehyde 37% and 3.7% must be prepared and used only in the chemical fume hood, using chemical resistant gloves. Waste must be kept in the fume hood and disposed of appropriately.

In the fume hood, tap the tube to loosen the pellet and resuspend each pellet in Amount500 µL of 3.7% formaldehyde. Mix well by pipetting up and down, ideally using a Rainin 1ml electronic pipette to obtain a single cell suspension and incubate at room temperate to fix for 10-20 minutes.

Centrifuge cells at Centrifigation300 x g, 00:03:00 using a minifuge inside the fume hood.

Carefully remove the supernatent (dispose of waste formaldehyde appropriately) and resuspend the cell pellet in Amount500 µL PBS and transfer to a 5ml BD Falcon Round Bottom Polystyrene Tube by pipetting through its filtered cap.
Note
At this stage, the fixed cell suspension can either be stored at Temperature4 °C overnight or analysed using flow cytometry immediately.