Mar 04, 2025

Public workspaceCulturing RBL-2H3 rat basophils

DOI
dx.doi.org/10.17504/protocols.io.14egn67qpl5d/v1
  • 1Arcadia Science
  • Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.14egn67qpl5d/v1
Protocol Citation: Justin Donnelly, Emily C.P. Weiss 2025. Culturing RBL-2H3 rat basophils. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn67qpl5d/v1
Manuscript citation:
Borges AL, Donnelly J, Morazan E, Rollins M. (2025). Compound 48/80 is toxic in HMC1.2 and RBL-2H3 cells. https://doi.org/10.57844/arcadia-3207-4695
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: May 10, 2024
Last Modified: March 14, 2025
Protocol Integer ID: 99551
Keywords: tissue culture, mast cell, basophil
Abstract
Guidelines for thawing, maintaining, subculturing, and freezing RBL-2H3 (rat basophil) line.
Materials
ReagentRBL-2H3 rat basophil cellsATCCCatalog #CRL-2256
ReagentT75 flasks for cell cultureThermofisher
ReagentMast Cell Degranulation Assay KitMerck MilliporeSigma (Sigma-Aldrich)Catalog #IMM001
ReagentEagles Minimum Essential MediumVWR InternationalCatalog #MSPP-302003
ReagentHeat-inactivated fetal bovine serumThermofisherCatalog #A3840101
Reagent70% ethanolVWR International
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
T25 flasks
ReagentIMDMGibco - Thermo FischerCatalog #12440053 ReagentPenStrepInvitrogen - Thermo Fisher Reagent1-ThioglycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6145
ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
PBS
15 mL centrifuge tubes
Protocol materials
Reagent70% ethanolVWR International (Avantor)
ReagentPenStrepInvitrogen - Thermo Fisher
Reagent1-ThioglycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6145
ReagentRBL-2H3 rat basophil cellsATCCCatalog #CRL-2256
ReagentT75 flasks for cell cultureThermofisher
ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
Reagent70% ethanolVWR International (Avantor)
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
ReagentMast Cell Degranulation Assay KitMerck MilliporeSigma (Sigma-Aldrich)Catalog #IMM001
ReagentEagles Minimum Essential MediumVWR International (Avantor)Catalog #MSPP-302003
ReagentHeat-inactivated fetal bovine serumThermofisherCatalog #A3840101
ReagentIMDMGibco - Thermo Fisher ScientificCatalog #12440053
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056
ReagentHeat-inactivated fetal bovine serumThermofisherCatalog #A3840101
ReagentEagles Minimum Essential MediumVWR International (Avantor)Catalog #MSPP-302003
ReagentPenStrepInvitrogen - Thermo Fisher
Thawing the RBL-2H3 rat basophil cell line
Thawing the RBL-2H3 rat basophil cell line
8m
8m
To make the complete culture medium for these cells, add ReagentHeat-inactivated fetal bovine serumThermofisherCatalog #A3840101 to a final concentration of Concentration15 % (v/v) in ReagentEagles Minimum Essential MediumVWR InternationalCatalog #MSPP-302003 . Add ReagentPenStrepInvitrogen - Thermo Fisher to a final concentration of 1×. Sterile-filter the media under strict aseptic conditions.

It's important to avoid excessive alkalinity of the medium during recovery of the cells. Prior to adding cells, you should place the culture vessel containing the complete growth medium into the incubator for at least Duration00:15:00 to allow the medium to reach Ph7.0 –Ph7.6 .

15m
The frozen cell culture should have been stored in liquid nitrogen and not at Temperature-70 °C , as storage at Temperature-70 °C will result in loss of viability. To use the cells, first thaw the vial by gentle agitation in a Temperature37 °C water or bead bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water (if using). Thawing should be rapid (approximately Duration00:02:00 for water bath, Duration00:05:00 for bead bath).

7m
Remove the vial from the water bath as soon as the contents are thawed, and decontaminate the outside of the vial by dipping in or spraying with Reagent70% ethanolVWR International . All of the operations from this point on should be carried out under strict aseptic conditions.

Working quickly to avoid loss of viability, transfer the vial contents to a centrifuge tube containing Amount9.0 mL complete culture medium and spin at approximately Centrifigation125 x g, 00:05:00 .

5m
Resuspend cell pellet with the recommended complete medium (see the specific batch information for the recommended dilution ratio) and dispense into a 75 cm2 culture flask.

Incubate the culture at Temperature37 °C in a suitable incubator with a 5% CO2 in air atmosphere.

Subcultivation
Subcultivation
10m
10m
Subculture the cells every 2–3 days. We recommend a subcultivation ratio of 1:4 to 1:8.

Remove and discard used culture medium.

Rinse the cell layer with ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056 or PBS to remove traces of serum.

Add Amount2.0 mL to Amount3.0 mL of ReagentTrypsin-EDTA (0.25%)Thermo Fisher ScientificCatalog #25200056 solution to the flask. Incubate cells at 37 °C until they detach — for RBL-2H3, this is usually within Duration00:10:00 .

10m
Add Amount6.0 mL to Amount8.0 mL of complete growth medium (≥ 2× the trypsin volume) and dislodge remaining cells in the flask by gently pipetting.

Add appropriate aliquots of the cell suspension to new culture vessels and incubate the culture at Temperature37 °C in a suitable incubator. We recommend 5% CO2 in air atmosphere.

Cryopreservation
Cryopreservation
8m
8m
Cryopreserve many aliquots of low-passage cells for future experiments.

Recover cells from flask as in 7.1–7.4.

Count cells. Prepare a mixture of Amount10 µL cells and Amount10 µL trypan blue, inverting cells gently before removing sample for counting. Mix well, then transfer Amount10 µL trypan-diluted cells to a Countess slide. Count using Countess to determine cell density and relative viability.

Freeze cells at ~1 million live cells in Amount1 mL cryo-media per vial. Ensure there are enough live cells for the desired number of aliquots. Centrifuge cells to be frozen at Centrifigation125 x g, 00:05:00 .

5m
Meanwhile, prepare cryo-media by diluting ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438 in complete growth medium to a final concentration of Concentration10 % (v/v) .

After centrifugation, aspirate supernatant. Resuspend cell pellet in cryo-media. Working quickly to avoid loss of viability, aliquot cells to pre-labeled screw-top cryotubes.

Place cells in a Mr. Frosty or similar container that controls freezing rate. Place container at Temperature-80 °C DurationOvernight .

10m
Transfer frozen cells to vapor-phase liquid nitrogen for long-term storage.

Protocol references