Q1
What are the recommendations for improving the purity of target cells for negative separation?
Here we have organized the following suggestions:
Good recovery and purity are based on high quality MNC preparation where the number of granulosa cells and platelets is low.
• Use the HulaMixerTM sample mixer (catalog number 15920D) in the mixing step to ensure that enough antibodies are bound to the target cells.
• The cells are well washed after the antibody binding step to remove unbound antibodies.
The 2X rosetting protocol increases cell purity without increasing the number of DynabeadsTM beads used in the separation process.
Q2
What is the trick to improve the final sorting efficiency/yield during the negative separation?
Here we have organized the following suggestions:
• Sample preparation: All separations must be performed in a single cell suspension.
· Make sure to use the correct number of cells, dilution factor and antibody mixture volume, DynabeadsTM magnetic beads and buffer system.
• Use the HulaMixerTM sample mixer (catalog number 15920D) in the mixing step to ensure good mixing in the separation operation.
• In the final step before touching the magnetic stand, the user must use a 1 mL tip to blow more than 10 times to fully resuspend the cells/beads. This operation can help the user capture the retained cells of interest.
• PBS containing the separation and wash buffer should be free of Ca2+ and Mg2+ to avoid complement activation or aggregation of the cells.
Q3
When the monocyte-negative separation is performed, the cell aggregation is encountered. What should I do?
Platelet activation often causes trouble in the isolation of monocytes, since activated platelets bind to monocytes and produce an unfavorable condition of monocyte and platelet aggregation. There are several types of factors that can trigger platelet activation effects, such as temperature changes, mechanical factors (ie, shear forces), exposure to the anticoagulant heparin (usually present in the sample tube), and the like. To avoid platelet activation (and subsequent monocyte aggregation), here are some general recommendations to follow:
• Switch to other anticoagulants other than heparin (eg ACD, EDTA or sodium citrate).
• Blood samples should be stored at room temperature until MNC is prepared and the MNC preparation process itself should be performed at room temperature.
• Samples should be handled gently to minimize mechanical shear. If possible, manually inject the blood sample using a syringe/needle instead of the VacutainerTM tube, as the vacuum conditions in such sample tubes usually result in the co-activation of platelets and monocytes.
Sodium citrate is known to "stabilize" platelets and avoid their activation, and includes - in addition to citrate buffer as an anticoagulant - theophylline, adenosine and dipyridamole.
· Make sure to prepare MNC according to our protocol. These steps are designed to achieve low platelet content of MNC.
Q4
The received InvitrogenTM DETACHaBEADTM magnetic beads look turbid, is this normal?
Yes, the turbid appearance of DETACHaBEADTM is normal, and protein precipitation may occur due to the high protein content. This is not bacterial contamination and does not affect the recovery and viability of the isolated and released cells. Mix the solution thoroughly before use.
Q5
Is there any skill to increase cell yield for the use of DETACHaBEADTM products?
Here we have organized the following suggestions:
• Mix the DETACHaBEADTM solution thoroughly before use.
• The most critical parameter for achieving good yields is the mixing effect of DETACHaBEADTM solution and sample during incubation. We recommend that the user use a mixer or rotator (such as the HulaMixerTM Sample Mixer (Cat. No. 15920D)) that maintains the movement of the tube and keeps the sample at the bottom of the tube to prevent the beads from drying out.
• Reduce incubation time during cell separation (lower affinity increases release efficiency).
• After incubation with the DETACHaBEADTM product, place the magnetic beads-bound cells up and down at least 10 times before placing them on the magnetic frame adsorption operation to remove as much of the magnetic beads as possible from the cells. Too intense blowing will reduce the vitality of the cells.
• Avoid unnecessary delays during operation.
Q6
How can I increase cell yield during the process of isolating cells using any of the positive isolation kits?
Here we have organized the following suggestions:
• The sample preparation process is very important. All separations must be performed in a single cell suspension. For operations that separate a type of cell (such as a monocyte) directly from a whole blood sample, we recommend that you wash the blood sample before separation to remove the interfering factors.
• A suitable mixer must be used for all incubation procedures that specify a “mixing†operation. Mixers capable of providing both tilt and rotation functions or reverse mixing functions (such as the HulaMixerTM sample mixer (catalog number 15920D)) can be used.
• If using the FlowCompTM kit, add antibodies (indirect methods) to the cells before adding the beads. Excess antibodies should be removed by washing before adding the beads.
• After incubation with the dissociation reagent, we recommend that you use a 1 mL tip to blow the sample more than 10 times before placing the sample on the magnetic stand to fully resuspend the sample.
• The user must use the recommended separation buffer during cell separation. Ca2+ and Mg2+ should not be contained in PBS because divalent cations cause complement activation and cell aggregation.
• Cell aggregation occurs in the sample while severely reducing the yield and purity of cell separation.
Q7
Are there techniques to improve cell release efficiency during the use of the Invitrogen DynabeadsTM FlowcompTM Cell Separation Kit?
Here we have organized the following suggestions:
• Since the binding between DSB-X biotin-streptavidin is gradually increased over time; therefore, do not extend the release step to more than the time specified in the protocol. In some cases, shorter incubation times result in higher yields.
• After incubation with the dissociation reagent, we recommend that you use a 1 mL tip to blow the sample more than 10 times before placing the sample on the magnetic stand to fully resuspend the sample.
Q8
What should I do if I can't find a positive isolation kit for my cell type?
The best option is to use the DynabeadsTM FlowCompTM Flexi kit with a suitable target antibody for cell separation. The Flexi kit contains some of the reagents needed for separation and release operations, including the DSB-X biotinylation kit for biotinylation of your antibodies, modified streptavidin-coated FLowCompTM DynabeadsTM Magnetic Beads and FlowCompTM Release Buffer.
Q9
Is there a general skill to improve the efficiency of the CELLLectionTM release step?
Here we have organized the following suggestions:
• Make sure that the pH of RPMI + 1% FBS is not too high: DNase I is the most efficient between pH 7.0 and 7.4 (RPMI exposure to the atmosphere will cause an increase in pH and the pH indicator will turn purple).
• Do not use RPMI+10% FCS during DNase I treatment. Application of 10% FBS would result in lower cell yields than 1% FBS (possibly due to DNase I inhibitors contained in some batches of FBS).
• Make sure that the buffer contains the Mg2+/Ca2+ required for DNase activity.
· DNase I must be treated gently. Stirring the DNase solution vigorously reduces enzyme activity. Do not vortex the DNase solution.
· When recovering a lower number of cells, we recommend that you pre-coat the tube with RPMI + 1% FBS medium to reduce cell loss (the cells are very sticky and easy to attach to the tube wall during sorting) .
DNase I has good activity at 20 ° C. However, we recommend that the user preheat RPMI + 1% FBS to 37 ° C before the DNase I treatment step, because the room temperature of different laboratories is not the same.
• When cells are incubated with DNase release buffer, it is important to thoroughly blow the magnetic bead-cell mixture prior to separation using a magnetic stand, which provides mechanical force to break the DNA connection. Failure to carefully blow the cells will reduce the rate of cell growth.
Q10
Can you provide some tips for improving your yield during the application of the CELLLectionTM Biotin Binding Kit or the CELLLectionTM Universal Mouse lgG Kit?
Here we have organized the following suggestions:
• Titrate/optimize the amount of target antibody during the coating of CELLectionTM DynabeadsTM magnetic beads.
• If the number of cells of interest is very low, or the target antigen concentration on the cell surface is very low, indirect methods are recommended.
• Use >1 x 10E7 beads/mL sample (>25 μL).
• Wash whole blood samples before use.
• To ensure the highest efficiency of cell release, never vortex the DNase solution during the lysing of lyophilized DNase.
• Preheat the freshly prepared RPMI/1% FCS to 37 °C during cell release.
• Ensure that the pH of the buffer is between 7.0 and 7.4 to achieve the desired DNase I activity. Higher pH values ​​inhibit DNase activity.
• RPMI should contain sufficient amounts of Mg2+ to help DNase to function.
• When cells are incubated with DNase release buffer, it is important to thoroughly blow the magnetic bead-cell mixture prior to separation using a magnetic stand, which provides mechanical force to break the DNA connection. Failure to carefully blow the cells will reduce the rate of cell growth.
Q11
In the process of expanding T cells, why do cells die after activation with DynabeadsTM magnetic beads?
Provide some tips for maintaining optimal cell growth conditions:
• It is important to maintain optimal growth density of cells at 0.5–1.5 x 10E6 cells/mL. When the cell density exceeds 1.5-2.5 x 10E6 cells/mL, stimulation is required again.
• Re-stimulate every 8-12 days according to the magnetic bead: cell ratio provided in the product manual.
• Please follow the correct magnetic bead: cell ratio for each product provided in the manual for activation. Excessive magnetic beads are used to inhibit cell activation/amplification.
· Using optimized cell culture medium, add:
· Growth factors (IL-2, IL-7, IL-15, or other cytokines)
· Growth-promoting additives (eg human serum/FBS, L-glutamine/Glutamax, free radical scavenger (10 mM N-acetylcysteine))
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