Product Introduction
Peripheral blood mononuclear cell (PBMC)
(For research only)
PBMC is separated from the spleen by grinding, filtration and other methods. The PBMC in the spleen and in the whole blood differs slightly in type and ratio. The spleen is the largest blood storage organ in the body, and there are slight differences in cell types and cell ratios between PBMCs in the spleen and PBMCs in whole blood, but this does not affect the experiment.
Ⅰ. Reagents and Materials
- PBMC(Cat# LV-PBMC001/2/3)
- Medium (complete RPMI1640, see chart)
Component |
Usage |
Incomplete solution of RPMI1640 |
500ml |
L-Glutamine(L-Glu) |
2mM |
2-Mercaptoethanol(2-ME) |
50μM |
Penicillin |
100μg/ml |
Streptomycin |
100μg/ml |
FCS (inactivated at 56 °C for 30 min) |
50ml |
-37°C Thermostatic water bath
-Biosafety cabinet
-15 ml Centrifuge tube
-37°C/5% CO2 Incubator
-Wide mouth dropper and wide mouth pipette tip
-Pipette
-75% Alcohol
Ⅱ. Safe Guarding
When recovering cells, the experimenter should wear disposable masks, latex gloves, goggles, etc., and conduct relevant operations in the biosafety cabinet to ensure the safety and biosafety of the experimenter.
Ⅲ. Cell Recovery and Plating
1. In the biosafety cabinet, add 10ml complete RPMI1640 medium containing 300 mm glucose to a 15ml centrifuge tube, preheat the 37℃ constant temperature water bath for 10min, then transfer to the biosafety cabinet; put the complete RPMI1640 medium into a 37℃ constant temperature water bath to fully preheat.
2. Quickly transfer the frozen PBMCs from the refrigerated position to a thermostat water bath of 37°C. Immerse them as much water as possible at 37°C and shake horizontally, but make sure that the cap of the freezing tube is kept above the water.
3. Defrost the freezing tube until the cells inside are just completely dissolved into liquid, about 60-120 seconds.
4. Sterilize the freezing tube with 75% alcohol and transfer it to a biosafety cabinet.
5. Gently pipette the thawed cells twice. Carefully mix the cells and transfer dropwise to a centrifuge tube containing pre-warmed recovery medium (Note: If cells remain on the tip of the cryopreservation tube, aspirate 1 ml of recovery medium to wash the cryopreservation tube and the pipette tip. Then mix it in the medium of cryopreservation tube), slightly invert the centrifuge tube 2-3 times to mix evenly.
6. Centrifuge of 650×g for 5min, ascending to 7 and descending to 4.
7. Discard the supernatant (dump it to retain the residual liquid at the bottom to minimize cell loss), add 3 ml of complete RPMI1640 medium to re-suspend the cells and count with trypan blue staining.
8. Plate the medium according to the counting results and the experimental requirements.
9.Incubate in a 37 °C/5% CO2 incubator.
IV. Customer Service
If you find any quality problems with the product, please collect the original data and contact the company's salesmen or technical support at the first time. The company ensure after-sales service. Every laboratory has different conditions, different operating habits, different proficiency, and objective factors in experimental failure. If the operation is not strictly in accordance with the instructions or exceeds the time limit of after-sales, the company does not do after-sales. Please understand and support us.
Validity period and original data provided:
Recovery problems: Within 24 hours of resuscitation, trypan blue staining or PI staining should be provided.
Pollution problems: Within 96 hours of resuscitation, microscope photographs of differences should be provided.
Purity issues: Within one month, immunofluorescence or flow cytometry results should be provided.V. Contact Number
Tel: 0755-28284050
Technical Support: 19902901483 (Dr. Zhou)