Nov 05, 2019

Public workspaceCreation of HBSS Wash Buffer | Brusko Lab

DOI
dx.doi.org/10.17504/protocols.io.8xbhxin
  • 1University of Florida - Diabetes Institute
Brusko Laboratory
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DOI: dx.doi.org/10.17504/protocols.io.8xbhxin
Protocol Citation: Brusko Laboratory, Matthew Brown, Thinzar Pe Myint 2019. Creation of HBSS Wash Buffer | Brusko Lab. protocols.io https://dx.doi.org/10.17504/protocols.io.8xbhxin
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: In development
We are still developing and optimizing this protocol
Created: October 31, 2019
Last Modified: November 05, 2019
Protocol Integer ID: 29379
Materials
MATERIALS
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150
ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
STEP MATERIALS
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150
Protocol materials
ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150
ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095
ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150
Before start
Obtain all materials and apparatus required for this procedure. Transfer into a biological safety cabinet using aseptic techniques.
Open a sterile 1 liter (L) Rapid-Flow Sterile Disposable Filter Unit with a 0.2 micrometer (µm) PES Membrane.
Equipment
Rapid-Flow Sterile Disposable Filter Unit with PES Membrane
NAME
Filtration System
TYPE
Nalgene
BRAND
5670020
SKU
https://www.fishersci.com/shop/products/thermo-scientific-nalgene-rapid-flow-sterile-disposable-filter-units-1000ml-pore-size-0-20-m-membrane-90mm/0974103
LINK


Remove the lid of the filter flask and add Amount940 mL of (1x) HBSS to the filter.
ReagentHBSS (1x)Gibco - Thermo Fisher ScientificCatalog #14175-095


Add a single Amount50 mL aliquot of FBS and a Amount10 mL aliquot of Penicillin/Streptomycin (Pen/Strep) to the HBSS in the filter system

ReagentFetal Bovine Serum (FBS)Genesee ScientificCatalog #25-550

ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL

Add Amount10 µL of Concentration0.5 Molarity (M) EDTA to the solution.

ReagentEDTA (0.5 Molar)Boston BioproductsCatalog #BM-150

Process the final solution through the 0.2 µm PES membrane using a positive-pressure system.
Once the solution has been filtered, remove the filter from the flask by unscrewing the filter and dispose of the filter into biohazardous waste.

Safety information
Since this procedure makes use of penicillin and streptomycin, any disposable products that come into contact with these antibiotics should be disposed of in biohazards waste to prevent the creation of antibiotic-resistant bacteria.

Remove the flask lid from the secondary container and securely screw the cap onto the flask. Label the flask containing the sterile HBSS Wash Buffer with the following information: "HBSS with FBS, EDTA, Pen/Strep" as well as your name and the date.
Store the HBSS Wash Buffer in Temperature4 °C .