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Negative Isolation

Here are some recommendations:

  • Good recovery and purity depends on a high-quality MNC preparation with low numbers of granulocytes and platelets.
  • Use a HulaMixer™ Sample Mixer (Cat. No. 15920D) during mixing steps to make sure there is sufficient antibody binding to the target cells. 
  • Wash the cells well after the antibody binding step to remove unbound antibodies.
  • The 2X rosetting protocol can increase purity without increasing the amount of Dynabeads™ magnetic beads used in the isolation.

Here are some recommendations:

  • Sample preparation: All isolations must be performed on single-cell suspensions. 
  • Make sure to use the correct cell number, dilutions, and volumes of antibody mix, Dynabeads™ magnetic beads, and buffers.
  • Use an Invitrogen™ HulaMixer™ Sample Mixer (Cat. No. 15920D) during mixing steps to make sure there is good mixing during the isolation procedure. 
  • In the last step, the cell/bead pellet must be resuspended well by pipetting >10 times using a 1 mL pipette prior to adding to the magnet. This is to capture possible trapped target cells in the rosettes. 
  • PBS used in making the isolation and washing buffer should be Ca2+ and Mg2+ free to avoid complement activation and aggregation of cells.

Platelet activation often constitutes a problem when isolating monocytes, as activated platelets bind to monocytes and produce unfavorable aggregates of monocytes and platelets. There are several factors that can trigger platelet activation, such as temperature changes, mechanical factors (e.g., shear), exposure to the anticoagulant heparin (often present in sampling tubes), etc. In order to avoid platelet activation (and subsequent monocyte aggregation), there are a few general recommendations that should be followed: 

  • Use anti-coagulants (such as ACD, EDTA, or sodium citrate) other than heparin. 
  • Blood samples should be kept at room temperature until MNC preparation, and the MNC preparation itself should be performed at room temperature. 
  • Samples should be treated gently to minimize mechanical shearing. If possible, draw blood manually using a syringe/needle rather than a Vacutainer™ tube, as the vacuum in such sampling tubes could often lead to activation of both platelets and monocytes. 
  • Sodium citrate is known to "stabilize" platelets and prevent activation, and contains—besides a buffered citrate solution as anticoagulant—theophylline, adenosine and dipyridamole. 
  • Be sure to follow our protocol for MNC preparation, as this is designed to obtain MNC with low platelet numbers.
Positive Isolation

Yes, the cloudy appearance is normal for DETACHaBEAD™ beads because the protein content is very high and the protein can precipitate. This is not bacterial contamination and does not affect the recovery or viability of the isolated and released cells. Mix the solution thoroughly before use.

Here are some recommendations:

  • Mix the DETACHaBEAD™ solution thoroughly before use. 
  • The most critical parameter for a good yield is mixing of the beads with the sample during the incubation step. We recommend using a mixer or rotator (e.g., HulaMixer™ Sample Mixer (Cat. No. 15920D)) that keeps the tube continuously in motion, but in such a way that the sample stays in the bottom part of the tube to avoid drying out of the beads
  • Reduce the incubation time during cell isolation (lower binding affinity can increase release efficiency). 
  • After incubation with DETACHaBEAD™ beads, pipette the bead-bound cells up and down at least 10 times before applying to the magnet to get as many beads off the cells as possible. Too vigorous pipetting can lower the viability. 
  • Avoid unnecessary delays during the protocol.

Here are some suggestions:

  • Sample preparation is very important. All isolations must be performed on single-cell suspensions. For isolation of certain cell types (e.g., monocytes) directly from whole blood, we recommend that you wash the blood before isolation to remove interfering factors in the sample.
  • An appropriate mixer must be used for all incubations where ‘mixing’ is specified. Any mixer providing either tilting and rotation or end-over-end mixing can be used (e.g., HulaMixer™ Sample Mixer (Cat. No. 15920D)). 
  • If using FlowComp™ kits where the antibodies are added to the cells before adding the beads (indirect method), excess antibodies should be removed by washing prior to adding the beads. 
  • After incubation with the releasing agent, we recommend that you re-suspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet. 
  • The recommended isolation buffer must be used for cell isolations. The PBS must be Ca2+ and Mg2+ free as these divalent ions can lead to activation of complement and aggregation of cells. 
  • Aggregation of cells in the sample can severely reduce both yield and purity of the isolation.

Here are some suggestions:

  • Since the DSB-X biotin -streptavidin bond becomes stronger over time; do not increase the time of the release step longer than stated in the protocol. In some instances a shorter incubation time can lead to higher yield. 
  • After incubation with the releasing agent, we recommend that you resuspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet.

The best choice is to use the Dynabeads™ FlowComp™ Flexi Kit in combination with a suitable target antibody for cell isolation. The Flexi kit contains both a DSB-X biotinylation kit for biotinylation of your antibody, the modified streptavidin-coated FlowComp™ Dynabeads™ magnetic beads, and the FlowComp™ Release Buffer required for performing isolation and release.

Here are some suggestions:

  • Make sure that pH of the RPMI + 1% FBS is not too high: DNase I works best between pH 7.0–7.4 (O2 exposure of RPMI will result in increased pH and the pH-indicator will turn blue).
  • Do not use RPMI + 10% FCS during DNase I treatment. 10% FBS gives a lower cell yield than 1% FBS (might be caused by DNase I inhibitory factors in some batches of FBS).
  • Make sure that the buffer contains Mg2+/Ca2+ required for DNase activity.
  • DNase I must be treated gently. Vigorously stirring of the DNase solution can reduce the enzyme activity. Never vortex the DNAse solution.
  • When selecting low numbers of cells, we recommend that you pre-coat tubes with RPMI + 10% FBS to reduce cell loss (cells are sticky and will easily attach to the tube wall during selection).
  • DNase I has good activity at 20°C, however, we recommend pre-warming the RPMI + 1 %FBS to 37°C before starting DNase I treatment because ‘room temperature’ varies from lab to lab.
  • After cells are incubated with DNase Releasing Buffer it is essential that the bead-cell complexes are thoroughly pipetted before the magnetic separation to provide mechanical disruption to the DNA linker. Failure to pipette the cells will affect cell yield.

Here are some suggestions:

  • Titrate/optimize the target antibody amount when coating the CELLection™ Dynabeads™ magnetic beads.
  • If the target cell number is low or the target antigen concentration on the cell surface is low, use the indirect technique.
  • Always use >1 x 10E7 beads per milliliter sample (>25 μL).
  • Wash whole blood before use.
  • To ensure the most efficient cell release, never vortex DNase when resuspending freeze-dried DNAse.
  • For cell release, use fresh RPMI/1% FCS pre-warmed to 37°C.
  • Check the pH of the RPMI. It should be 7.0–7.4. Higher pH will inhibit DNase activity.
  • RPMI should contain sufficient Mg2+ for DNase activity. 
  • After cells are incubated with DNase Releasing Buffer it is essential that the bead-cell complexes are thoroughly pipetted before the magnetic separation to provide mechanical disruption to the DNA linker. Failure to pipette the cells will affect cell yield.
Cell Activation and Expansion

Here are a few tips to keep the optimal cell culture growth conditions:

  • It is important to keep the cell density at 0.5– 1.5 x 10E6 cells/mL for optimal growth. When the cell density exceeds 1.5–2.5 x 10E6 cells/mL, re-stimulation is required.
  • Re-stimulate every 8–12 days with bead:cell ratio according to the product manual. 
  • Activate with the correct bead:cell ratio for each specific product according to the manual. Too many beads can inhibit cell activation/proliferation.
  • Use optimized cell culture media, add;
    • Growth factors (IL-2, IL-7, IL-15, or other cytokines) 
    • Growth enhancing supplements (e.g., human serum/FBS, L-glutamine/glutamax, radical scavenger (10 mM N-acetyl cysteine)

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