FireGene Tumor Dissociation Kit - Optimized for Tumor Microenvironment

Product FAQ

1.    Q: Is this kit only suitable for fresh tumor tissue? Is it effective for cryopreserved and resuscitated tumor tissue? Can it be used for non-solid tumor samples (e.g., hematological tumor cells)?

A: The kit is mainly suitable for fresh solid tumor tissues (such as lung cancer, breast cancer, and liver cancer tissues). It should be used with caution for cryopreserved and resuscitated tumor tissues — the cryopreservation process may cause structural damage to tumor cells, and the cell viability after dissociation will be 20%-30% lower than that of fresh tissues. Trypan blue staining is required for pre-quality inspection (viability ≥60% to be usable). It is not suitable for non-solid tumor samples. Hematological tumors (such as leukemia and lymphoma) are mainly composed of suspended cells, which do not require tissue dissociation and can be processed directly with a cell sorting kit. The enzymolysis system of this kit is not compatible with suspended tumor cells and may cause massive cell death.

2.    Q: The instruction manual requires cutting 200mg of fresh tumor tissue. Will insufficient tissue quantity (e.g., 80mg) or excessive tissue quantity (e.g., 300mg) affect the dissociation effect? How to adjust the operation?

A: Yes, it will affect the effect. ① Insufficient tissue quantity (<150mg): The dissociation solution is relatively excessive, and the high enzyme concentration easily leads to over-digestion of tumor cells, resulting in a viability decrease of more than 40%. It is recommended to combine multiple 80mg tissue samples (to a total weight of 180-200mg) and add the dissociation solution according to the conventional dosage to ensure the ratio of enzyme to tissue is appropriate. ② Excessive tissue quantity (>250mg): The space in the 5mL centrifuge tube is limited, so the tissue cannot be fully dispersed, and some areas may have incomplete enzymolysis, leaving a large amount of tissue debris. The tissue needs to be processed in two tubes, with each tube containing 180-220mg of tissue and corresponding to 240μL of dissociation solution + 2760μL of RPMI 1640 medium, to avoid the impact of tissue crowding on enzymolysis efficiency.

3.    Q: The digestion time in Step 3 is 0.5-2 hours. How to determine the initial digestion time for different types of tumor tissues (e.g., lung cancer, breast cancer, liver cancer)? What consequences will occur if the digestion time is insufficient or excessive?

A: The initial digestion time needs to be adjusted according to the density of the tumor tissue: ① Tumors with low density (such as some lung cancers and solid foci of lymphoma): The initial digestion time is 0.5-1 hour. Such tissues have low fiber content and are easy to enzymolyze. ② Tumors with high density (such as breast cancer and liver cancer): The initial digestion time is 1-1.5 hours. Such tissues contain more collagen fibers and require a longer time for decomposition. Insufficient digestion time: The tissue is not fully dissociated, and a large number of clusters of more than 10 cells can be seen under the microscope, with a single-cell yield of less than 50%. Excessive digestion time: Tumor cells are excessively damaged, with viability reduced to less than 50%, and cell debris is prone to appear, which interferes with the preparation of subsequent single-cell sequencing samples. Dynamic adjustment of the termination time through quality inspection every 20 minutes is required.

4.    Q: Steps 5 and 6 require filtration with a 70μm cell sieve and rinsing the centrifuge tube 3 times to collect a total of 12mL of filtrate. What impact will omitting one rinsing step or replacing with a 50μm/100μm sieve have?

A: Omitting one rinsing step will result in the loss of approximately 1/3 of residual tumor cells, reducing the final cell yield by 25%-30%. Tumor cells are easily adsorbed on the inner wall of the centrifuge tube, and 3 rinsing steps are the key to ensuring full cell recovery. A 50μm or 100μm sieve cannot be used as a replacement: The pore size of the 50μm sieve is too small and will retain some single tumor cells (e.g., small cell lung cancer cells have a diameter of about 8-12μm; although they can pass through, the sieve is easily blocked by fiber debris, leading to filtration difficulties and even cell extrusion to reduce viability). The pore size of the 100μm sieve is too large and cannot effectively filter undigested tissue fragments. These fragments will enter subsequent steps along with cells, which not only interferes with the accuracy of cell counting but also may block the single-cell sequencing chip.

5.    Q: The instruction manual mentions that "DMEM medium can replace RPMI 1640 medium". After replacement, is it necessary to adjust centrifugation parameters or digestion time? Is there any difference in the impact of the two media on the viability of tumor cells?

A: There is no need to adjust centrifugation parameters (still 4℃, 300×g for 5 minutes) or digestion time after replacement. Both DMEM and RPMI 1640 are common basal media for mammalian cells. Although they differ in glucose and amino acid content, both can provide a suitable osmotic pressure (280-320mOsm/kg) and pH (7.2-7.4) for tumor tissue dissociation, with no impact on enzymolysis efficiency. The impact on the viability of tumor cells is minimal: Experimental data shows that the difference in cell viability after dissociating breast cancer tissue with the two media is ≤5%. The medium can be selected based on the existing inventory in the laboratory without deliberate replacement. If tumor cell culture is required in subsequent steps, it is recommended to prioritize the medium consistent with the culture system to reduce the cost of cell adaptation.

6.    Q: Step 12 mentions that "BA3311 Red Blood Cell Lysis Buffer can be used to remove red blood cells". After which step should this operation be performed? What should be noted during lysis to avoid damaging tumor cells?

A: This operation should be performed after Step 10 (after two washes) and before Step 11 (before resuspension). Specific operation: After discarding the supernatant in Step 10, add 1mL of BA3311 Red Blood Cell Lysis Buffer, incubate at 4℃ for 5 minutes (to avoid damaging tumor cells due to room-temperature incubation), then centrifuge at 4℃, 300×g for 5 minutes, discard the supernatant, and resuspend with 5mL of PBS containing 5% FBS (add an additional washing step), then proceed to Step 11. Precautions: ① The lysis time should not exceed 8 minutes; the 4℃ environment can reduce the toxicity of the lysis buffer to tumor cells. ② If there are too many red blood cells (such as in blood-rich tumor tissues), lysis can be repeated once, but an additional PBS washing step is required to avoid residual lysis buffer affecting subsequent experiments (e.g., cell capture efficiency in single-cell sequencing).

7.    Q: The tumor tissue dissociation solution needs to be stored at -20℃ with a validity period of two years. If the ice pack melts during transportation and the reagent is left at 4℃ for 3 hours, can it still be used? What impact does repeated freezing and thawing have?

A: It can still be used after being left at 4℃ for 3 hours, but it must be immediately returned to -20℃ and fully mixed before use. The enzymes (such as collagenase and neutral protease) in the dissociation solution lose ≤10% of their activity when placed at 4℃ for a short period (≤3 hours), which does not affect the dissociation effect. Repeated freezing and thawing will lead to a significant decrease in enzyme activity: Each freeze-thaw cycle reduces enzyme activity by 12%-18%; after more than 3 freeze-thaw cycles, the activity is less than 50%, which cannot effectively decompose the extracellular matrix of tumor tissue, resulting in a large number of tissue blocks after dissociation and even failure to obtain a single-cell suspension. It is recommended that after receiving the kit, aliquot the 2×1.25mL dissociation solution into 250μL/tube (each tube corresponds to one experimental dose), seal it, store at -20℃, and take one tube for each experiment to avoid repeated freezing and thawing.

8.    Q: After centrifugation in Steps 8 and 9, the supernatant needs to be discarded. If many suspended tumor cells are still found in the supernatant after centrifugation, how to deal with it? Can it be improved by extending the centrifugation time or increasing the rotation speed?

A: The presence of suspended tumor cells in the supernatant is mostly caused by improper centrifugation parameters or insufficient cell precipitation. Treatment method: Collect the supernatant and re-centrifuge at 4℃, 350×g for 8 minutes (increase the rotation speed by 50×g and extend the time by 3 minutes compared with the original parameters). If there are still a small number of suspended cells after centrifugation, collect the supernatant again and repeat the centrifugation once to ensure full cell recovery. It is not recommended to excessively increase the rotation speed (>400×g) or extend the time (>10 minutes): Excessively high rotation speed will squeeze tumor cells, leading to cell membrane rupture and a 20%-25% decrease in viability; an excessively long time has little impact on cell viability but will increase the experimental time. Effective improvement can be achieved by operating according to the adjusted parameters mentioned above.

9.    Q: After quality control, it is required to "carry out subsequent experiments immediately". If subsequent experiments cannot be conducted immediately, can the prepared tumor cell suspension be stored for a short period? What are the restrictions on storage conditions and time?

A: Short-term storage is possible under the conditions of sealed storage in a 4℃ refrigerator for no more than 1 hour, with repeated shaking avoided. During storage, adjust the cell concentration to 1×10⁶-1×10⁷ cells/mL using PBS containing 5% FBS, and place it in a low-adhesion centrifuge tube to prevent cell loss due to adhesion. After 1 hour, the viability of tumor cells will decrease significantly (10%-12% per hour), and cell aggregation is prone to occur; if stored for more than 3 hours, the cell viability may be lower than 50%, making it unsuitable for single-cell sequencing or cell culture. Before use, re-quality inspection is required, and only cells with viability ≥65% and single-cell ratio ≥75% can be used. At the same time, gently pipette 5-8 times to disperse slightly aggregated cells.