Cell Guide: OCI-LY3 (Human Diffuse Large B-Cell Lymphoma Cell) Culture Protocol
Basic Cell InformationCell Name: OCI-LY3 (Human Diffuse Large B-Cell Lymphoma Cell)
Cell Aliases: OCI-LY3; OCI-ly3; OCI-LY-3; Oci-Ly-3; OCI-Ly 3; OCILY-3; OCI-Ly03; OCI Ly3; OCILY3; Ly3; LY3
Cell Catalog Number: VWT1B140
Growth Characteristics: Suspension
Cell Introduction: The OCI-LY3 cell line was established in 1983 from a bone marrow sample of a 52-year-old male with B-cell non-Hodgkin lymphoma (B-NHL, diffuse large B-cell lymphoma, DLBCL, stage 4B) at relapse. These cells are activated B lymphocytes. The literature describes OCI-LY3 cells as EBV-negative and lacking the typical t(8;14) translocation.
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Important Notices for OCI-LY3 Cell Culture
· OCI-LY3 cells grow predominantly in suspension, forming grape-like clusters. Large cell clumps require gentle pipetting to break into smaller clumps to prevent cell death within the clump center due to nutrient insufficiency. Small clumps do not need to be disrupted. Unless required by the experiment, it is not recommended to dissociate the cells into single cells.
· OCI-LY3 cells have high serum requirements; high-quality serum should be used for culture, with a serum concentration of 20%.
· As culture time increases, some cells may attach and spread on the culture surface. During passaging, gently pipette the surface to detach these cells. The detached cells can be passaged normally, while cells that remain attached do not need to be collected.
· This cell line exhibits density-dependent culture characteristics; the cell seeding density should not be too low.
OCI-LY3 Cell Medium Renewal
Partial Renew
1. Prepare the required culture medium, centrifuge tubes, sterile pipette tips, etc. (Pre-warm the culture medium).
2. Let the culture flask stand for 5-10 minutes to allow cells to settle (observe cell settlement visually).
3. Keep the pipette tip close to the liquid surface and carefully aspirate half of the medium into a centrifuge tube.
4. Centrifuge at 900-1000 rpm for 3 minutes. If cells are present in the centrifuge tube, resuspend them in fresh culture medium and return the suspension to the original flask.
5. Add fresh culture medium to the original flask to replace the aspirated half-volume.
Centrifuge Method
1. Prepare the required culture medium, centrifuge tubes, sterile pipette tips, etc. (Pre-warm the culture medium).
2. Transfer the entire cell suspension to a centrifuge tube.
3. Centrifuge at 900-1000 rpm for 3 minutes.
4. Add an appropriate volume of fresh culture medium to the culture flask.
5. After centrifugation, discard the supernatant, gently resuspend the cell pellet in a small volume of fresh culture medium, and inoculate the cell suspension back into the culture flask. Mix well and place the flask back into the incubator.
OCI-LY3 Cell Subculture Methods
Centrifugation Method
1. Prepare the required culture medium, centrifuge tubes, sterile pipette tips, etc. (Pre-warm the culture medium).
2. Using a pipette, transfer the cell suspension from the culture flask to a centrifuge tube.
3. Centrifuge at 900-1000 rpm for 3 minutes to pellet the cells.
4. Add an appropriate volume of fresh culture medium to the culture flask (e.g., 10 mL for a T25 flask, 20 mL for a T75 flask).
5. After centrifugation, discard the supernatant. Add an appropriate volume of fresh culture medium, gently resuspend the cell pellet by pipetting to break clumps, and evenly distribute the cell suspension into new culture flasks. Gently mix by swirling and place the flasks back into the incubator.
Direct Splitting Method
1. Gently pipette the cell suspension to break clumps into small aggregates and mix well.
2. Using a pipette, aspirate the cell suspension from the culture flask and distribute equal portions into new culture flasks. Add an appropriate volume of fresh culture medium to each new flask, gently mix, and place the flasks back into the incubator.
Tips:
· Pay attention to keeping pipetting force gentle and minimize pipetting frequency to avoid mechanical damage to cells.
· Recommended subculture ratio: 1:2
OCI-LY3 Cell Cryopreservation Method
1. Prepare the required culture medium, PBS, serum, DMSO, centrifuge tubes, sterile pipette tips, etc. (Reagents should be pre-warmed).
· Tip: If the culture medium has turned yellow before cryopreservation, perform a medium change and culture the cells statically for about 4 hours to allow cell viability to stabilize before cryopreserving.
2. Based on the number of cells collected, prepare the corresponding volume of cryopreservation medium and the required number of cryovials. Label each cryovial with the cell name, passage number, cryopreservation date, etc. Recommended cryopreservation medium formulation: 90% FBS + 10% DMSO. Recommended cryopreservation density: 3 million cells/mL/cryovial.
· Tip: Low cryopreservation density will lead to poor cell viability upon thawing.
3. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 3 minutes to pellet the cells.
4. After centrifugation, discard the supernatant. Add the prepared cryopreservation medium to gently resuspend the cell pellet. Dispense the cell suspension into the cryovials. Pay attention to pipetting gently to avoid generating bubbles.
· Tip: After adding the cryopreservation medium and resuspending the cells, proceed to freeze the cells promptly to minimize DMSO toxicity.
5. Place the cryovials into a controlled-rate freezing container (e.g., "Mr. Frosty"), then transfer the container to a -80°C freezer for gradual cooling and freezing.
6. The next day (16-24 hours later), thaw one vial to check the freezing efficacy. Once confirmed satisfactory, transfer the cryopreserved cells to liquid nitrogen for long-term storage. Do not keep cells at -80°C for more than 3 days.
OCI-LY3 Cell Thawing Method
1. Pre-warm a water bath to 37°C. Warm the culture medium to room temperature or 37°C. Set the centrifuge speed.
2. Retrieve the cryovial from liquid nitrogen or the -80°C freezer. Immediately place it into the 37°C water bath and shake rapidly until completely thawed. The thawing time should not exceed 2 minutes.
· Tips:
· If the liquid nitrogen or freezer is far from the cell culture room, transport the cells on dry ice or an ice pack.
· Protect the cryovial from direct contact with water by wrapping it with a disposable PE glove during water bath thawing to prevent contamination.
3. Centrifuge the thawed cryovial at 900-1000 rpm for 2 minutes. Meanwhile, add an appropriate volume of fresh culture medium to the culture flask.
4. After centrifugation, discard the supernatant. Gently resuspend the cell pellet in 1 mL of fresh culture medium (a serum concentration increased to 20% is recommended). After gently pipetting to dissociate cells, inoculate the cell suspension into the prepared culture flask.
5. Gently swirl the flask using a cross or figure-eight motion to mix the cells evenly, then place it into the incubator.
· Tip: Complete the entire cell thawing procedure as quickly as possible, ideally within 5-10 minutes.
How to handle excessive cell debris during culture?
1. Black intracellular particles: These likely represent extracellular secretory vesicles and organelle refraction, an inherent characteristic of the cells. They cannot be eliminated or altered. Most cell lines exhibit this phenomenon, though the degree varies. Culture system inadequacy, nutrient deficiency, or osmotic pressure abnormalities can increase intracellular particles. It is recommended to use appropriate culture conditions.
2. Black extracellular particles: These are mostly cell debris and metabolic byproducts. A low-speed centrifugation (e.g., 800 rpm, 3 minutes) can remove some debris. A small amount of debris does not affect cell growth, and frequent centrifugation is not recommended.
