Mar 04, 2025

Public workspaceCulturing HMC1.2 human mast cells

DOI
dx.doi.org/10.17504/protocols.io.261ger8xwl47/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.261ger8xwl47/v1
Protocol Citation: Justin Donnelly, Emily C.P. Weiss 2025. Culturing HMC1.2 human mast cells. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger8xwl47/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: February 03, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 119506
Keywords: tissue culture, mast cell, basophil
Abstract
Guidelines for thawing, maintaining, subculturing, and freezing the HMC1.2 (human mast cell) 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
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
Step 8.4
Reagent70% ethanolVWR International (Avantor)
Step 3
ReagentHeat-inactivated fetal bovine serumThermofisherCatalog #A3840101
Step 1
Reagent1-ThioglycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6145
Step 1
ReagentPenStrepInvitrogen - Thermo Fisher
Step 1
ReagentIMDMGibco - Thermo Fisher ScientificCatalog #12440053
Step 1
Thawing the HMC1.2 human mast cell line
Thawing the HMC1.2 human mast 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 Concentration10 % (v/v) , Reagent1-ThioglycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6145 to a final concentration of Concentration1.2 millimolar (mM) , and ReagentPenStrepInvitrogen - Thermo Fisher to a final concentration of 1× in ReagentIMDMGibco - Thermo FischerCatalog #12440053 . Sterile-filter the media under strict aseptic conditions.

You should have kept the frozen cell culture 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 it in or spraying it with Reagent70% ethanolVWR International . Carry out all operations from this point on 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 Centrifigation300 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 25 cm2 culture flask.

Incubate the culture at Temperature37 °C in a suitable incubator with a 5% CO2 in air atmosphere.
Subcultivation
Subcultivation
Subculture the cells every 2–3 days when cell density reaches 1.0–1.5 million live cells/mL. The recommended seeding density for subculturing is 250,000 live cells/mL.

Transfer cells to a 15 mL tube. Dislodge any cells from 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 with a 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.

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.

Cells will be frozen 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 Centrifigation300 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