Home Protocols Protocol for Luna® Cell Ready Lysis Module (NEB #E3032)

Protocol for Luna® Cell Ready Lysis Module (NEB #E3032)

Before Use

  • Prepare cells in advance (various cell types such as adherent, suspension, or cryopreserved can be used) and ensure cells are intact before lysis.
  • Consider testing several cell dilutions to determine the appropriate input range to use for lysis and RT-qPCR. Although a standard curve is not required for high throughput screening experiments, it is still recommended.
  • It is important to use only a monolayer of cells in well-plate formats. We recommend using Surface Areas and Guide for
    Recommended Medium Volumes     from Corning® as a starting point for preparing cell cultures.
  • Ensure that all components are thawed and mixed prior to use. Once thawed, place on ice prior to use. Workflow using Luna Cell Ready One-Step RT-qPCR Kit.

Workflow using Luna Cell Ready One-Step RT-qPCR Kit.

Part I. Cell Lysate Preparation using Luna Cell Ready Lysis Module

Step 1. Processing Cells

Cell Resuspension Solution (CRS) is recommended for resuspending/diluting cells to reduce RNA damage during the handling process. When only a fraction of the whole sample is to be used (e.g., adherent cells grown in vessels other than 24/48/96/384 well plates, suspension cells, or cryopreserved cells), you should resuspend with CRS to protect your samples’ RNA during processing. CRS is not required for adherent cells in 24/48/96/384 well plates. 

Prepare Cell Resuspension Solution (CRS) by diluting Luna Cell Ready RNA Protection Reagent (25X) to 1X with cold PBS (e.g. for each sample, mix 2 μl Luna Cell Ready RNA Protection Reagent with 48 μl of 1X PBS to make 50 μl Cell Resuspension Solution). 

Adherent cells in 24/48/96/384 well plates Adherent cells grown in other vessels Suspension cells Cryopresered cells
  1. Remove all cell culture media.
  2. Rinse briefly with cold PBS and aspirate PBS*.
  3. Store on ice and proceed to lysis
    within 10 mins.
 Detach cells using common sub-culturing techniques.
  		  — Quickly thaw the
cell stock at 37°C.
  1. Transfer the desirable volume of cells and spin down the cell pellets. Carefully remove the supernatant.
  2. Rinse briefly with cold 1X PBS and aspirate PBS*.
  3. Resuspend cells with CRS (up to 20,000 cells/μl). Store on ice and proceed to lysis within 10 mins.

* For high throughput screening (e.g., adherent cells in 96 wells or 384 well plates), cell wash is optional if medium removal can be completed via a plate flipping step.


Step 2. Prepare Cell Lysis Mix

  1. Thaw Luna Cell Ready Lysis Buffer and Luna Cell Ready Stop Solution at room temperature, then place on ice with all other components. After thawing completely, briefly mix each component by inversion.

  2. Prepare Cell Lysis Mix of all components, adding the Luna Cell Ready Protease immediately before use.
    Mix thoroughly by pipetting gently. Centrifuge briefly to collect the solution to the bottom of the tube and store on ice. For best results, the Cell Lysis Mix should be used immediately (within 15 minutes).

    Component

    40 μl Cell Lysis Mix

    Final Concentration

    Luna Cell Ready Lysis Buffer (2X)

    25 μl

    1X

    DNase I (RNase-free) (10X)

    5 μl

    1X

    Luna Cell Ready RNA Protection Reagent (25X)

    2 μl

    1X

    Luna Cell Ready Protease (25X)

    2 μl

    1X

    Nuclease-free Water

    6 μl

    Up to 2,000 cells per μl lysis reaction is recommended. In a typical 50 μl lysis reaction, 100,000 cells can be lysed.

 

Step 3. Cell Lysis

For lysis using cells resuspended from adherent cells grown in vessels other than 24/48/96/384 well plates, suspension cells, or cryopreserved cells: Mix up to 5 μl of cells resuspended in CRS with the Cell Lysis Mix to a final volume of 45 μl. Gently pipet up and down 6 times. Incubate the lysis reaction at 37°C for 10 min.*

For lysis of adherent cells in 24/48/96/384 well plates: Aliquot an appropriate volume of Cell Lysis Mix into each well as indicated in the following table and incubate the reaction at 37°C for 10 min.*

For most efficient lysis, automatic shaking is recommended for cell densities higher than 200 cells/μl.

Culture Plate

Cell Lysis Mix Per Well

24 well

160 μl

48 well

80 μl

96 well

40 μl

384 well

8 μl

*Note: Lysis at room temperature is an option if cell density is less than 200 cells/μl in the lysate.

 

Step 4. Lysis Termination

Add 10X Luna Cell Ready Stop Solution to a final concentration of 1X and mix well by pipetting up and down 6 times. Centrifuge briefly to collect the solution to the bottom of the tube. Incubate at 25°C for 5 min.

For 96 well plates, we found that adding 5 μl of Luna Cell Ready Stop Solution (10X) to the 40 μl lysis solution (± 10% cells and residual wash buffer) was sufficient for complete lysis.

Cell lysates can be stored on ice for up to five hours, at -20°C for up to five days and at -80°C for long term storage. Cell lysate is stable for up to five freeze and thaw procedures.