Confining liquid and application thereof
A technology of blocking liquid and solution, which is applied in the direction of instruments, analytical materials, particles and sedimentation analysis, etc., can solve the problems of perishable, poor sealing effect of blocking liquid, etc., and achieve the effect of not easy to deteriorate, saving experimental cost, and accurate results
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Embodiment 1
[0043] This example is the induction of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes
[0044]1. HiPSC-U1 culture method: freeze-thaw Matrigel from -80°C to 4°C overnight, aliquot Matrigel the next day and add 100 μl Matrigel to 10ml DMEM / F-12 for culture in a 15ml centrifuge tube, store Store in the refrigerator at 4°C for later use. Every day, replace each well with new medium: 4ml TeSR-E8 medium until the number of hiPSCs reaches about 75%.
[0045] 2. On the 0th day of differentiation, discard the old medium, replace and add 4ml (12μM CHIR99021+RPMI / B-27minus insulin) medium to each well, and return the multi-well plate to 37°C, 5% CO 2 Incubate in the incubator for 24h.
[0046] 3. On the first day of differentiation, discard the old medium, add 4ml of RPMI / B-27 medium (without insulin) to each well, and return the multi-well plate to 37°C, 5% CO 2 Incubate in the incubator for 24h.
[0047] 4. On the third day of differentiation, 72 hours after ad...
Embodiment 2
[0051] This embodiment is the immunofluorescent staining of cardiomyocytes in Example 1
[0052] (1) Digestion:
[0053] Aspirate off the old medium and wash the differentiated cells twice with 2 ml of PBS per well. Aspirate the PBS, add 2ml (0.25% (wt / vol) trypsin-EDTA) dissociating enzyme to dissociate the cells, and digest at 37°C for 5min in a 5% incubator.
[0054] (2) filter cells:
[0055] Cells were singulated by pipetting 5-10 times with a 1 ml pipette. The pipetted single cell suspension was then transferred to a 15ml centrifuge tube containing 4ml RPMI 20, and the cells were naturally filtered by gravity with a 70μm cell sieve.
[0056] (4) Counting:
[0057] Take 10 μl of the cell suspension and count the cells with a hemocytometer; and centrifuge the cell suspension at a speed of 1000 r / min for 4 min at room temperature. After centrifugation, the supernatant was discarded using a 1 ml pipette.
[0058] (5) Cell fixation:
[0059] After the old medium was di...
Embodiment 3
[0070] In this example, flow cytometric analysis was performed on the cardiomyocytes after immunofluorescence staining in Example 2 and Comparative Example 1.
[0071] The cardiomyocytes incubated in Example 2 and Comparative Example 1 were washed twice with 2 ml FlowBuffer-2 respectively; the cell pellet was resuspended in 50 μl FlowBuffer-1, and transferred to a flowing round bottom tube. Place the flow tubes on ice and perform flow cytometric analysis with a FACS Calibur. The result is as Figure 1~6 .
[0072] Depend on Figure 1~3 and Figure 4~6 Comparative analysis shows that: in comparative example 1, the number of cells obtained by blocking liquid injection is relatively small, and the fluorescence detection value is low, so the amount of cells required for the experiment will deviate from the theoretical value of the experiment; in example 2, the number of cells obtained by blocking liquid injection is more than expected. There is little difference between the ex...
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