Cell recovery solution

Manufactured by Corning

Sourced in United States, Japan, Italy, United Kingdom

Cell Recovery Solution is a laboratory product designed for the gentle dissociation and recovery of adherent cells from cell culture surfaces. It is a buffered solution that aids in the detachment of cells without compromising their viability or functionality.

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Recovery solution (10 citations) Ice-cold cell recovery solution (5 citations) Matrisperse Cell Recovery Solution (5 citations) Matrigel with Cell Recovery Solution (2 citations) Cold cell recovery solution (2 citations)

380 protocols using cell recovery solution

Matrigel Embedded Cell Retrieval

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Corning^® Cell Recovery Solution enabled us to retrieve cells from Matrigel^®. Matrigel^®-embedded samples were placed at 4°C for 30 min allowing Matrigel^® to be liquified. All samples were transferred into new tubes with existent growth media and centrifuged at 1,200 rpm for 3 min at 4°C, then the supernatant was removed leaving only the Matrigel^® layer including cells. An appropriate amount of Cell Recovery Solution (CORNING, Cat #354253) was next added to the Matrigel^®/cell mix (the amount of the solution was recommended to be ≧2x that of Matrigel volume), which was then incubated at 4°C overnight. Cells were centrifuged at 1,200 rpm for 3 min at 4°C, and supernatant was aspirated. 1 mL of cold (4°C) PBS was added to the remaining cell pellet, mixed gently, then transferred into a microtube and centrifuged at 5,000 rpm for 3 min. After discarding the supernatant, cells were washed with cold PBS. Finally, cells were centrifuged at 15,000 rpm for 15 min, the supernatant discarded, and purified cell pellet was collected.

Tahara S., Sharma S., de Faria F.C., Sarchet P., Tomasello L., Rentsch S., Karna R., Calore F, & Pollock R.E. (2024). Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research. Frontiers in Cell and Developmental Biology, 12, 1362696.

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Comprehensive T Cell Infiltration Analysis

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RencaHA tdTomato cells were resuspended at a concentration of 1 × 10⁵ cells/ml, mixed with Matrigel (Corning) at 4 °C, seeded in a 24-well plate at a final concentration of 500 cells per Matrigel dome, and left to solidify for 10 min at 37 °C. 2 ml cell medium was added to each well and cells incubated at 37 °C for 11 days. Each Matrigel dome was washed twice in PBS and incubated for 30 min with 1 ml of Cell Recovery Solution (Corning). Spheroids were collected in a 15-ml Falcon tube and pulsed with K^dHA peptide at a final concentration of 2 μg/ml for 1 h. Pulsed spheroids were re-embedded in Matrigel together with 5 × 10⁵ primed CL4 CTL per Matrigel dome. Matrigel domes were dissolved for analysis of spheroid-infiltrating T cells after 16 h: Spheroids were washed twice in PBS and incubated with 1 ml of Cell Recovery Solution (Corning). Spheroids were collected, washed through a 70 μm sieve, and then disaggregated to retrieve T cells in 500 μl of imaging buffer for immediate FACS sorting.

Edmunds G.L., Wong C.C., Ambler R., Milodowski E.J., Alamir H., Cross S.J., Galea G., Wülfing C, & Morgan D.J. (2022). Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization. Communications Biology, 5, 9.

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Generation and Isolation of Tumor-Infiltrating Lymphocytes

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RencaWT mCherry cells were resuspended at a concentration of 1 x 10⁵ cells/ml, mixed with Matrigel (Corning) at 4°C, seeded in a 24-well plate at a final concentration of 500 cells/blob, and left to solidify for 10 min at 37°C. Cell medium (2 ml) was added to each well and incubated at 37°C for 11 days. Each blob was washed twice in PBS and incubated for 30 min with 1 ml of Cell Recovery Solution (Corning). Spheroids were collected in a 15-ml Falcon tube and pulsed with K^dHA peptide at a final concentration of 2 μg/mL for 1 hour. Pulsed spheroids were re-embedded in Matrigel together with 5 x 10⁵ primed Clone 4 CTLs per blob. The following day, blobs were washed twice in PBS and incubated with 1 ml of Cell Recovery Solution (Corning). Spheroids were collected, washed through a 70-μm sieve, and then disaggregated to retrieve SILs in 500 μL of imaging buffer for immediate FACS sorting.

Ambler R., Edmunds G.L., Tan S.L., Cirillo S., Pernes J.I., Ruan X., Huete-Carrasco J., Wong C.C., Lu J., Ward J., Toti G., Hedges A.J., Dovedi S.J., Murphy R.F., Morgan D.J, & Wülfing C. (2020). PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and target tumor cells within the tumor. Science signaling, 13(649), eaau4518.

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Immunostaining of Organoid Cultures

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For immunohistochemistry, organoids were washed from the BME R using Cell recovery solution (Corning), fixed overnight in 4% PFA and embedded in paraffin blocks. 5 mm-tick sections were processed for H&E staining and immunohistochemistry using Ki67 antibody (Abcam). For whole mount immunofluorescence staining, organoids were isolated from the BME R using Cell recovery solution (Corning), fixed overnight in 4% PFA, permeablilized and blocked by incubation in Triton X-100 0.2% and 5% normal donkey serum (Jackson Laboratories) in PBS for 1h at RT. Primary antibodies were incubated at 4 C overnight. After washing with PBS 1X, conjugated secondary antibodies were added for 1h at RT. DAPI was used to counterstain nuclei. Phalloidin (Sigma) was used for Actin staining. Organoids were mounted and imaged on an Sp8 confocal microscope (Leica). Images were processed using Photoshop CS4 or ImageJ software. Imaris software was used for images 3D-reconstruction.

Artegiani B., van Voorthuijsen L., Lindeboom R.G.H., Seinstra D., Heo I., Tapia P., López-Iglesias C., Postrach D., Dayton T., Oka R., Hu H., van Boxtel R., van Es J.H., Offerhaus J., Peters P.J., van Rheenen J., Vermeulen M, & Clevers H. (2019). Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids. Cell stem cell, 24(6).

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Organoid Protein Expression Analysis

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1,500 organoids treated or not with 100 nM 4OT for 7 d were harvested using 1 ml Corning Cell Recovery Solution (354253; Corning) per ∼60 µl BME gel. After centrifugation, they were lysed in 100 µl RIPA buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 5 mM EDTA, 50 mM NaF, and 1 mM PMSF supplemented with Roche 1X Halt Protease inhibitor cocktail), incubated for 1 h at 4°C, and sonicated using a Bioruptor PLUS (Diagenode) for 30 cycles of 30 s spaced by 30 s of cooling down. Total protein concentration in lysates was determined using Pierce BCA Protein Assay Kit (23227; Thermo Scientific). The protein concentration in the lysates was 1.74 µg/µl. Proteins were fractionated in 10% polyacrylamide gel and blotted on polyvinylidene difluoride membrane. Milk was used as a blocking agent.
Membranes were incubated with a Rabbit anti GORASP2 (Rich, against aa 232–454), at dilution 1/1,000; a gift from M. Bekier (University of Michigan, Ann Arbor, MI; Xiang and Wang, 2010 (link)); and a mouse monoclonal antibody to E-cadherin (610182 at 1/5,000; BD Biosciences).
The visualization was performed after incubation with secondary antibodies coupled to HRP (NA931 and NA934; GE Healthcare) and Clarity Western ECL Substrate (Bio-Rad) using ImageQuant LAS4000 ECL (GE Healthcare).

Grond R., Veenendaal T., Duran J.M., Raote I., van Es J.H., Corstjens S., Delfgou L., El Haddouti B., Malhotra V, & Rabouille C. (2020). The function of GORASPs in Golgi apparatus organization in vivo. The Journal of Cell Biology, 219(9), e202004191.

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Myotube Harvest and Gene Expression Analysis

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Myotubes were harvested from microfluidic chambers using Corning Cell recovery solution (Corning, USA) for 10 min and left for 1 h on ice until Matrigel was dissolved. Total RNA was then extracted by pooling 2 devices and using an RNeasy kit (Qiagen, USA), according to the manufacturer’s guidelines. cDNA was then synthesized with SuperScript™ II reverse transcriptase (ThermoFisher Scientific) from 1 mg of RNA per sample. Real-time qPCR was run with Light cycler 480 SYBR Green Master (Roche, Spain) in a StepONEPlus light cycler (ThermoFisher Scientific). C_ts were calculated following the manufacturer’s instructions. Expression values are expressed as 2^-ΔΔCt. First C_ts were normalized versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, and then the relative amount was normalized against corresponding non-stimulated controls. Primer sequences used are described in Table 1.

Sala-Jarque J., Mesquida-Veny F., Badiola-Mateos M., Samitier J., Hervera A, & del Río J.A. (2020). Neuromuscular Activity Induces Paracrine Signaling and Triggers Axonal Regrowth after Injury in Microfluidic Lab-On-Chip Devices. Cells, 9(2), 302.

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Isolation and Culture of Fibroblast-HLMEC Co-cultures

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A mixture of FACS‐sorted Col1α1‐GFP mice fibroblasts and HLMECs was plated in a 1:1 solution of Matrigel Matrix (Corning, Cat# 354248) and endothelial cell growth basal medium (Lonza, Cat# 00190860) as detailed in Appendix S1. After 3 days, cells were removed from Matrigel with Corning Cell Recovery Solution (Corning, Cat#35425), and total RNA, cDNA synthesis, and qPCR analysis were performed.

Caporarello N., Meridew J.A., Aravamudhan A., Jones D.L., Austin S.A., Pham T.X., Haak A.J., Moo Choi K., Tan Q., Haresi A., Huang S.K., Katusic Z.S., Tschumperlin D.J, & Ligresti G. (2020). Vascular dysfunction in aged mice contributes to persistent lung fibrosis. Aging Cell, 19(8), e13196.

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Matrigel Removal and Organoid Harvesting

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Prior to fixation, or lysis, organoids were removed from Matrigel using Corning Matrigel Cell Recovery Solution (Corning, 354253). First, organoids were harvested into a 15 ml tube using a wide Pasteur pipette and washed with 10 ml of cold washing medium (Advanced DMEM/F12, 1X GlutaMax, 1 mM Hepes and Penicillin/Streptomycin). The 15 ml tube was inverted every 2 min for 10 min, followed by 5 min incubation on ice before organoids were spun 200 g at 4°C. This was repeated once and then Corning Cell Recovery Solution (Corning, 354253) was used to further remove the Matrigel (incubation on ice for 30 min with inversion once after 15 min). Organoids were washed with cold PBS, spun down at 200 g 4°C.

Nikolić M.Z., Caritg O., Jeng Q., Johnson J.A., Sun D., Howell K.J., Brady J.L., Laresgoiti U., Allen G., Butler R., Zilbauer M., Giangreco A, & Rawlins E.L. (2017). Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids. eLife, 6, e26575.

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Cell Growth Kinetics Measurement

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The growth of both MCF-7 and OVCAR-8 cell lines was measured for 14 days. 6 × 10⁵ cells per well were plated in both gradient and control 6-well plates. Cells were cultured as previously described, then harvested at days 1, 3, 5, 7, 10, and 14. Plates were removed from the incubator and the media aspirated. Corning^™ Cell Recovery Solution (200 μL, Corning Life Sciences, Bedford, MA) was added to each well and the plates were placed on a rocker at 4 °C for 1 h. The solution from each well was transferred to a separate collection tube, and 1 mL PBS 10× was used to rinse each well to collect the remaining cells. Cells were then counted using a Cellometer^™ Auto T4 (Nexelcom Biosciences, Lawrence, MA).

Wulftange W.J., Rose M.A., Garmendia-Cedillos M., da Silva D., Poprawski J.E., Srinivasachar D., Sullivan T., Lim L., Bliskovsky V.V., Hall M.D., Pohida T.J., Robey R.W., Morgan N.Y, & Gottesman M.M. (2019). Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression. Journal of cellular physiology, 234(11), 20608-20622.

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Biliary Organoid Dissociation Protocol

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Biliary organoids embedded in BME (EMB), 10% BME, and AAOs were dissociated into single cells. Briefly, domes with EMB organoids were dissolved in ice-cold Corning Cell Recovery solution (Corning) for 10 minutes. After washing with PBS, all organoid types were spun down and resuspended in 0.25% trypsin and incubated for 30 minutes at 37 °C. At the end of the incubation, organoids were mechanically dissociated by pipetting. Trypsin digestion was blocked by the addition of ice-cold resuspension buffer (2% FBS, 1.25 mM CaCl₂, 4 mM MgCl₂, 10 mM HEPES, 5 mM glucose in 1X HBSS), and cells were recovered by centrifugation. Cells in the resuspension buffer were filtered through a 35-µm filter (Corning), counted, and checked for viability using Countess II automatic cell counter (Thermo Fisher Scientific). Cells, at the desired concentration, were resuspended in PBS-0.04% BSA.

Fiorotto R., Mariotti V., Taleb S.A., Zehra S.A., Nguyen M., Amenduni M, & Strazzabosco M. (2023). Cell-matrix interactions control biliary organoid polarity, architecture, and differentiation. Hepatology Communications, 7(4), e0094.

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