KC-6421

293T-ADGRL1 Cell Line

62712

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Background of 293T-ADGRL1 Cell Line

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. This gene encodes a member of the latrophilin subfamily of G-protein coupled receptors (GPCR). Latrophilins may function in both cell adhesion and signal transduction. In experiments with non-human species, endogenous proteolytic cleavage within a cysteine-rich GPS (G-protein-coupled-receptor proteolysis site) domain resulted in two subunits (a large extracellular N-terminal cell adhesion subunit and a subunit with substantial similarity to the secretin/calcitonin family of GPCRs) being non-covalently bound at the cell membrane. Diseases associated with ADGRL1 include Developmental Delay, Behavioral Abnormalities, And Neuropsychiatric Disorders and Specific Learning Disability.

Specifications

Catalog Number	KC-6421
Cell Line Name	293T-ADGRL1 Cell Line
NCBI/UniProt Accession Number	O94910
Clone Number	5#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous human ADGRL1 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:5 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-ADGRL1 cell line was generated using a lentiviral vector expressing the human ADGRL1 sequence.

Characterization

Figure 1: Characterization of human ADGRL1 overexpression in the 293T-ADGRL1 stable clone using FACS.

Figure 2: Characterization of human ADGRL1 in the 293T-ADGRL1 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (DMEM+10% FBS +1μg/mL Puromycin) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:5 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Vitobello A, Mazel B, Lelianova VG, Zangrandi A, Petitto E, Suckling J, Salpietro V, Meyer R, Elbracht M, Kurth I, Eggermann T, Benlaouer O, Lall G, Tonevitsky AG, Scott DA, Chan KM, Rosenfeld JA, Nambot S, Safraou H, Bruel AL, Denommé-Pichon AS, Tran Mau-Them F, Philippe C, Duffourd Y, Guo H, Petersen AK, Granger L, Crunk A, Bayat A, Striano P, Zara F, Scala M, Thomas Q, Delahaye A, de Sainte Agathe JM, Buratti J, Kozlov SV, Faivre L, Thauvin-Robinet C, Ushkaryov Y. ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model. Am J Hum Genet. 2022 Aug 4;109(8):1436-1457. doi: 10.1016/j.ajhg.2022.06.011. Epub 2022 Jul 30. PMID: 35907405; PMCID: PMC9388395.
2. Dietzsch AN, Al-Hasani H, Altschmied J, Bottermann K, Brendler J, Haendeler J, Horn S, Kaczmarek I, Körner A, Krause K, Landgraf K, Le Duc D, Lehmann L, Lehr S, Pick S, Ricken A, Schnorr R, Schulz A, Strnadová M, Velluva A, Zabri H, Schöneberg T, Thor D, Prömel S. Dysfunction of the adhesion G protein-coupled receptor latrophilin 1 (ADGRL1/LPHN1) increases the risk of obesity. Signal Transduct Target Ther. 2024 Apr 26;9(1):103. doi: 10.1038/s41392-024-01810-7. PMID: 38664368; PMCID: PMC11045723.
3. Liang H, Tang LY, Ge HY, Chen MM, Lu SY, Zhang HX, Shen CL, Shen Y, Fei J, Wang ZG. Neuronal survival factor TAFA2 suppresses apoptosis through binding to ADGRL1 and activating cAMP/PKA/CREB/BCL2 signaling pathway. Life Sci. 2023 Dec 1;334:122241. doi: 10.1016/j.lfs.2023.122241. Epub 2023 Nov 7. PMID: 37944639.