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Protocol

Protocol

CodePlex Secretome

IsoPlexis Applications Team, IsoPlexis

Jul 22, 2021

Abstract

This protocol outlines the standard method for thawing and loading samples onto the CodePlex chip.

Introduction

For first time users, it is highly recommended to first practice loading with a CodePlex Training Chip, which is provided for new users with their first order Read MSDS documents of all materials prior to use. Laboratory workers should wear standard PPE, including disposable gloves, protective eyewear, and laboratory coats.

Reagents and Equipment

CodePlex Kit Components:

  • CodePlex Reagent Box (Store at 4°C)
    • 15 mL Tube A
    • 15 mL Tube B
    • 1.5 mL Tubes A/B: Cocktail A in Micro-Tube (Green Cap) and Cocktail B in Micro-Tube (Red Cap)
    • 50 mL Tubes containing Reagents 1, 2, 3, 4, 5, 6, 7, 8 1
    • Bag of Disposable Reagent Sippers
  • CodePlex Chip Set (Store at -20°C)
    • Boxes of CodePlex Chips (1, 2, 4, 6, or 8 chips)
  •  CodePlex Required Accessories (Store at Room Temperature)
    • Cover Tape (One per chip)
    • Cover Tape Applicator (One per kit)
    • P10 Filtered Pipette Tips (One rack for 1, 2 and 4 chips; two racks for 6 and 8 chips)*
    • CodePlex Calibration Chip
    • CodePlex Training Chip (new users only)

*Note: P10 filtered pipette tips provided as part of the CodePlex kit are not compatible with Rainin LTS pipettes. 


Procedure

Before Getting Started:

1. Important Precautions 

  • Read MSDS documents of all materials prior to use.
  • Working with Biohazardous Reagents: Please refer to your institute’s guidelines and obtain proper training to handle potentially biohazardous samples. It is also strongly recommended that any lab personnel handling human samples should be vaccinated against HBV if the individual does not have sufficient HBV antibody titer.
  • Additional precautions need to be taken when working with samples that potentially contain an EID agent:
    • Laboratory workers should wear standard PPE, including disposable gloves, protective eyewear, and laboratory coats.
    • Any procedure or process that cannot be conducted in the designated EID BSC should be performed while wearing gloves, gown, goggles and a fit tested N-95 mask.
    • Work surfaces should be decontaminated on completion of work with appropriate disinfectants. This includes any surface that potentially comes in contact with the specimen (centrifuge, microscope, etc.)
    • All liquid waste produced in the processes must be treated to a final concentration of 10% bleach prior to disposal.

2. Reagents to Be Prepared Before Starting

3. Guidelines for Sample Stability

IsoPlexis recommends the following general guidelines for storing your sample. This is not intended to be an allinclusive listing. Please refer to your institute’s guidelines for long term sample storage if applicable. 

1. Avoid repeated freeze-thaw cycles of samples as this may degrade, partially or fully, the sample quality. 

  • If a sample needs to be run multiple times, aliquot smaller volumes into single-use low protein binding tubes and thaw as needed.

2. For cell supernatant, store samples in a low protein binding tube. 

3. Cell supernatant continuously stored at -80°C should be stable for at least 1 year. 

  • If possible, arrange to run samples as soon as possible to minimize duration-based degradation.
  • Literature reports suggest stability for up to 2 years in most cases. However, IsoPlexis has not independently verified this information, and in general urges caution when running extremely old samples.

4. When collecting cell supernatants, be sure to save an aliquot of the same media batch used during supernatant generation for use as the Background Control. 

Protocol:

Day 1: Thawing and Loading

*Guidelines for Urine Samples for Analysis on CodePlex Secretome
Urine samples need to be processed prior to loading on the CodePlex Secretome chip utilizing the following method.

  1. After sample collection, centrifuge sample for 10 minutes at 2000 rcf at room temperature.
  2. Remove supernatant from particulate pellet formed by centrifugation.
  3. Sample may now be loaded onto CodePlex chips. It is recommended that the samples be run undiluted to maintain intensity of each cytokine signature. Samples may also be aliquoted and stored at -80°C for later analysis.
  4. Urine samples may be normalized using a creatinine assay per industry standard. This normalization is conducted separately from the CodePlex Secretome assay.

Methods:

1. Remove vacuum sealed bag containing CodePlex chips from -20ºC. CRITICAL: Chips must stay sealed until loading. 

2. Place CodePlex chips on a bench to thaw in the vacuum sealed bag at ambient temperature 60 to 75 minutes prior to opening the vacuum bag. 

3. Allow frozen samples to completely thaw at room temperature. TIP: Mix well by pipetting up and down prior to loading. Use a larger volume pipette (e.g., 100-1000 µL) to mix, depending on volume of sample. P10 pipette used to dispense sample into the chip will not provide adequate mixing for volumes greater than ~25 µL. 

4. Optimally, while chips and samples thaw, prepare CodePlex liquid reagents and attach all reagent tubes to IsoPlexis instrument. Refer to your instrument’s system guide for detailed instructions. 

5. Once thawed, remove CodePlex chips from vacuum sealed bag and place on a flat surface. CRITICAL: Keep protective blue film on bottom of chip

CRITICAL: Each well of the CodePlex chip must be loaded with sample or background control in numerical order and each well of a row must be filled before loading the wells of the next row. Wells 5, 6, 15, and 16 are labeled “B” and are designated for loading background controls, all other wells may be loaded with sample. Use only the Background Controls indicated in Table 9 and load all four Background wells with the same control fluid. All samples are loaded in duplicate wells and both wells are required to run the assay correctly. 

6. Using a P10 pipette, load 5.5 µL of Sample “a” into CodePlex well 1, firmly inserting the pipette tip into the well to ensure the pipette tip creates a seal around the well opening. Discard pipette tip. CRITICAL: Only dispense the sample to the first stop of the pipette to prevent bubbles from forming. DO NOT release the plunger. With the plunger still held at the first stop, wait for 2 seconds for the sample to load, then slowly remove the tip from the well to avoid disturbing the sample. 

Note: It is important to only use the P10 pipette tips supplied with the CodePlex kit as only certain pipette tips have been validated for use. Failure to do so can result in failure to create a seal between the pipette tip and the well opening. P10 pipette tips provided as part of the CodePlex kit are not compatible with Rainin LTS pipettes. 

7. Repeat step 6 for duplicate loading of Sample “a” into CodePlex well 2. CRITICAL: Use a new pipette tip for each well to avoid introducing air bubbles into the sample. 

8. Load 5.5 µL of Sample “b” into CodePlex wells 3 and 4, as described in the previous steps. 

9. Load 5.5 µL of the background control into well 5. 

Note: Wells 5, 6, 15, and 16 of the CodePlex chip are designated for loading background control and must not be loaded with sample. 

10. After loading wells 1 through 5, invert the CodePlex chip and inspect sample fill length through the glass slide on the bottom of the chip. If any samples are filled less than 75% of the length between the well inlet and first sample divider of the next row (Figure 2), lightly tap chip parallel to benchtop (slide side down) to promote sufficient sample filling. Inspect sample fill in between tapping and stop once each sample has loaded at least 75% of the well length. Alternatively, chip may be lightly tapped perpendicular to benchtop (barcode side down) to promote filling. 

CRITICAL: Tap CodePlex chip lightly. Excessive force can cause sample contamination into the adjacent wells. 

11. Load 5.5 µL of background control into well 6. 

12. Load remaining samples in duplicate into the remaining wells in order from well 7 to well 20, loading background controls into wells 15 and 16. CRITICAL: DO NOT load out of order. Loading out of order may result in sample cross-contamination. 

TIP: If you have less than 8 samples, you can leave the remaining wells blank. You must load all 4 background wells. You must load in order. 

TIP: Invert chip and inspect fill volume through the glass slide after each row of 5 wells is loaded to ensure each sample has filled at least 75% of the well length before loading the next row. 

TIP: Refer to Sample Loading Template (Appendix 1). 

13. After loading all samples and background controls in duplicate, gently invert chip to inspect sample loading through glass slide. As shown in Figure 2, liquid should cover at least 75% of the length of the sample chamber.

TIP: In the unlikely event that an individual sample well has not loaded adequately, insert, by hand, a clean P10 pipette tip into the underfilled well inlet. Invert the chip while maintaining a gentle hold of the inserted pipette tip. Using gloved fingertip, lightly apply pressure on the exposed end of the tip, while observing sample through glass slide, to promote filling until minimum fill length achieved. 

CRITICAL: There should be a visible sample divider between each sample in each row. In the event of sample loading errors and/or contamination between adjacent samples, as shown in Figure 3, affected wells should be noted on sample loading template and excluded from IsoSpeak analysis. 

14. Once the CodePlex chip has been inspected for proper sample loading, apply the Cover Tape: 

  • Peel off the clear liner of the Cover Tape completely, exposing the adhesive side of the tape.
  • Carefully align the Cover Tape to the top of the CodePlex chip, using the white rubber seals and outlined engravings on the chip as guides.
  • Place the Cover Tape down and use a finger to apply even pressure to smooth and seal the tape across the entire surface of the CodePlex chip.
  • Using the Cover Tape Applicator provided in the CodePlex Kit, apply moderate pressure across the Cover Tape to fully seal it to the chip. Slide the flat blade of applicator back and forth several times over each portion of the tape, first lengthwise (Figure 4, Top and Center) and then widthwise (Figure 4, Bottom). TIP: Slide the blade until it touches the rubber seals on each end. Slight indents can be seen over the well inlets when sufficient sealing pressure is applied. 

CRITICAL: Failure to properly seal the CodePlex chip with Cover Tape may result in sample leakage, loss of data, and IsoLight damage. DO NOT touch the center hole of the white rubber seals on either end of chip, as this may cause cross contamination in adjacent samples. 

15. Once the CodePlex chip has been loaded and Cover Tape has been applied, perform a final brief inspection of sample fill length and sample dividers between samples through the glass slide. 

Loading Chips in Instrument:

16. Select CodePlex Secretome from IsoPlexis instrument’s primary screen. Load CodePlex Calibration Chip provided with the CodePlex Kit into tray position 4 for IsoLight or tray position 2 for IsoSpark when prompted, with small screw facing the instrument and “CAL” label facing up as shown in Figure 6. The Calibration Chip contains standard curve data and enables the instrument’s system and IsoSpeak software to display concentrations in pg/mL.

17. Continue following on screen prompts, and after removing Calibration Chip, proceed to load CodePlex Secretome chips into instrument chip tray according to your instrument’s system guide. 

18. Remove the blue protective tape from the bottom surface of the chip. Immediately load CodePlex chip into the instrument with the white rubber seals facing up and with the small screw facing the instrument. Chip should securely mate with the magnet in the chip tray. Continue loading all chips into tray. CRITICAL: Handle CodePlex chips with care. Hold CodePlex chips by sides or barcode tab. DO NOT touch slide. DO NOT touch or apply pressure to the white rubber seals (inlet and outlet). DO NOT stack chips. 

19. Verify that all CodePlex liquid reagent tubes are securely attached to the instrument, prepared previously during chip thaw, and that the waste bottle is at least half empty (refer to your instrument’s system guide for details). 

20. Press “Close Chip Tray” and the instrument will start scanning the chip barcodes. The instrument will also verify that the Cover Tape has been properly applied. If the cover tape is not detected, the Software will display an error and allow the user to open the tray to fix tape application. 

21. Press “Start Assay” after all chips are scanned. Refer to your instrument’s system guide for additional details and tips as necessary.

 


Notes and Comments

To view the appendices or to view the original manuscript, download a PDF of the protocol here.

For troubleshooting, contact Support at 475-221-8402 & support@isoplexis.com.


References

IsoPlexis


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